Functionally distinct sodium channels in ventricular epicardial and ...

Am J Physiol Heart Circ Physiol 295: H154 –H162, 2008. First published May 2, 2008; doi:10.1152/ajpheart.01327.2007.

Functionally distinct sodium channels in ventricular epicardial and endocardial cells contribute to a greater sensitivity of the epicardium to electrical depression J. M. Cordeiro, M. Mazza, R. Goodrow, N. Ulahannan, C. Antzelevitch, and J. M. Di Diego Masonic Medical Research Laboratory, Utica, New York Submitted 12 November 2007; accepted in final form 29 April 2008

Cordeiro JM, Mazza M, Goodrow R, Ulahannan N, Antzelevitch C, Di Diego JM. Functionally distinct sodium channels in ventricular epicardial and endocardial cells contribute to a greater sensitivity of the epicardium to electrical depression. Am J Physiol Heart Circ Physiol 295: H154 –H162, 2008. First published May 2, 2008; doi:10.1152/ajpheart.01327.2007.—A greater depression of the action potential (AP) of the ventricular epicardium (Epi) versus endocardium (Endo) is readily observed in experimental models of acute ischemia and Brugada syndrome. Endo and Epi differences in transient outward K⫹ current and/or ATP-sensitive K⫹ channel current are believed to contribute to the differential response. The present study tested the hypothesis that the greater sensitivity of Epi is due in part to its functionally distinct early fast Na⫹ current (INa). APs were recorded from isolated Epi and Endo tissue slices and coronaryperfused wedge preparations before and after exposures to elevated extracellular K⫹ concentration ([K⫹]o; 6 –12 mM). INa was recorded from Epi and Endo myocytes using whole cell patch-clamp techniques. In tissue slices, increasing [K⫹]o to 12 mM reduced Vmax to 51.1 ⫾ 5.3% and 26.8 ⫾ 9.6% of control in Endo (n ⫽ 9) and Epi (n ⫽ 14), respectively (P ⬍ 0.05). In wedge preparations (n ⫽ 12), the increase in [K⫹]o caused selective depression of Epi APs and transmural conduction slowing and block. INa density was not significantly different between Epi (n ⫽ 14) and Endo (n ⫽ 15) cells, but Epi cells displayed a more negative half-inactivation voltage [⫺83.6 ⫾ 0.1 and ⫺75.5 ⫾ 0.3 mV for Epi (n ⫽ 16) and Endo (n ⫽ 16), respectively, P ⬍ 0.05]. Our data suggest that reduced INa availability in ventricular Epi may contribute to its greater sensitivity to electrical depression and thus may contribute to the R-ST segment changes observed under a variety of clinical conditions including acute myocardial ischemia, severe hyperkalemia, and Brugada syndrome. Brugada syndrome; acute ischemia; severe hyperkalemia

that acute myocardial ischemia (AMI) induces a greater electrical depression of ventricular epicardial (Epi) versus endocardial (Endo) tissues (9, 12, 15, 19, 31, 32). The presence of a prominent transient outward K⫹ current (Ito)-mediated spike and dome morphology in Epi (9, 25) has been suggested to be in part responsible for the differential response as a result of the development of all-ornone repolarization at the end of phase 1 in Epi (loss of the dome) but not in Endo (4, 7, 8, 21, 26, 27). This differential response may, at least in part, underlie the ST segment elevation observed in the surface ECG, particularly when the right ventricle (RV) is involved (11). Our data also indicated that a markedly delayed transmural conduction or transmural conduction block may underlie the R-ST segment and T wave changes encountered during acute ischemia (9). Other studies IT HAS LONG BEEN RECOGNIZED

Address for reprint requests and other correspondence: J. M. Di Diego, Masonic Medical Research Laboratory, 2150 Bleecker St., Utica, NY 13504 (e-mail: [email protected]). H154

(13, 22) have also shown a greater Epi sensitivity to ATPsensitive K⫹ channel current (IK,ATP) activation and suggested that this effect may contribute to its greater vulnerability to ischemic conditions. The accumulation of K⫹ in the extracellular space is an important component of the ischemic myocardium. In the clinical setting, severe hyperkalemia (ⱖ8 mM) often leads to ECG patterns of R-ST segment and T wave changes referred to as pseudo-AMI (29, 34), diffuse monophasic AP (MAP)-like ECG (17), or Brugada-like ECG (24). The underlying cellular bases for these ECG changes, as well as those observed in clinical AMI, are incompletely understood. Moreover, Na⫹ channel inhibition induced by an intravenous infusion of Na⫹ channel blockers is used to unmask or amplify the Brugada syndrome ECG pattern (ST segment elevation in the right precordial leads, V1–V3) (14). Brugada syndrome has been linked to loss of function mutations in SCN5A (5). The proposed underlying basis for the manifestation of this genetic disease is, in essence, similar to that of acute ischemia (i.e., the ST segment elevation is secondary to accentuation of the RV Epi AP notch and/or loss of the AP dome) (1, 10, 11, 27, 39). Given that the conditions mentioned above (ischemia, severe hyperkalemia, Na⫹ channel inhibition, and mutations in SCN5A linked to the Brugada syndrome) have in common a reduction in Na⫹ channel availability, we tested the hypothesis that the greater sensitivity of Epi versus Endo to conditions that reduce Na⫹ channel availability is in part due to its intrinsically distinct early fast Na⫹ current (INa). We tested this hypothesis by exposing superfused Endo and Epi canine ventricular tissue slices and arterially perfused ventricular wedge preparations to elevated extracellular K⫹ concentration ([K⫹]o) and by measuring early INa in isolated ventricular Epi and Endo myocytes. MATERIALS AND METHODS

Ventricular Endo and Epi preparations. This investigation conformed to the National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996) and was approved by the Animal Care and Use Committee of the Masonic Medical Research Laboratory. Adult mongrel dogs of either sex were used to perform all experimental series. Animals were anticoagulated with heparin and anesthetized with pentobarbital (30 –35 mg/kg iv). The chest was opened via a left thoracotomy, and the heart excised, placed in a cardioplegic solution (4°C Tyrode solution with 12 mM [K⫹]o), and transported to a dissection tray. Endo and Epi slices were 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.

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INa AND EPICARDIAL SENSITIVITY TO ELECTRICAL DEPRESSION

shaved (Davol Simon Dermatome Power Handle with cutting head no. 3295, Cranston, RI) from the basal portions of the RV and left ventricle (LV) (dimensions: 5–10 mm width and 10 –20 mm length, 0.5–1.0 mm thick). Isolated tissues were superfused with Tyrode solution (see below) equilibrated with 95% O2-5% CO2 at 37 ⫾ 0.5°C. AP recordings were obtained referenced to ground using glass microelectrodes (2.7 M KCl, 10 –25 M⍀ direct current resistance) connected to a high-input impedance amplification system (World Precision Instruments, New Haven, CT) at a sampling rate of 40 kHz. Stimulation was applied through a pair of thin silver electrodes. Stimuli were bipolar rectangular pulses of 3-ms duration and twice threshold intensity at basic cycle lengths (BCLs) ranging from 300 to 800 ms. Ventricular wedge preparations. Transmural wedges with dimensions of up to 3 ⫻ 2 ⫻ 1.5 cm (LV wedge) or 2 ⫻ 1.5 ⫻ 0.9 cm (RV wedge) were dissected from the base of the RV and LV. During the cannulation procedure, preparations were initially arterially perfused with cardioplegic solution. Subsequently, wedge preparations were placed in a tissue bath and perfused with Tyrode solution of the following composition (in mM): 129 NaCl, 4 KCl, 0.9 NaH2PO4, 20 NaHCO3, 1.8 CaCl2, 0.5 MgSO4, and 5.5 glucose, buffered with 95% O2 and 5% CO2 (37 ⫾ 0.5°C). The perfusate was delivered at a constant pressure (45–50 mmHg). A transmural pseudo-ECG (ECG) was recorded using two Ag/AgCl half cells placed ⬃1 cm from Epi (⫹) and Endo (⫺) surfaces of the preparation and along the same axis as the transmembrane recordings. APs were simultaneously recorded from Epi (surface) and Endo (⬃2–3 mm from the Endo surface) using floating glass microelectrodes. Basic pacing was applied to the Endo surface (BCLs ⫽ 300 –2,000 ms). Both Endo and Epi pacing were used to measure the excitability threshold (ET) at all experimental conditions and cycle lengths studied. ECG and AP signals were amplified and/or digitized and analyzed using Spike 2 for Windows (Cambridge Electronic Design, Cambridge, UK). The term “R-ST segment” refers to the part of the wedge ECG between the onset of the R wave and the beginning of the T wave. R-ST segment changes resembling MAPs were used in this study to describe the ECG changes attending the high-[K⫹]o experiments in which a dramatic widening of the R wave (particularly at fast rates) merges with, and becomes indistinguishable from, pure ST segment changes. Similarly, as we stated earlier, in the clinical setting of severe hyperkalemia, ECG patterns of R-ST segment changes resembling MAPs are commonly described as mentioned and referred to as pseudo-AMIs, diffuse MAP-like ECGs, or Brugada-like ECGs (17, 24, 29, 34). Isolated myocyte preparation. Myocytes from Epi and Endo regions were prepared from canine hearts using previously described techniques (6, 25). In brief, male and female adult mongrel dogs were anesthetized with pentobarbital sodium (35 mg/kg iv), and their hearts were rapidly removed and placed in nominally Ca2⫹-free Tyrode solution. A wedge consisting of the LV free wall supplied by a descending branch of the circumflex artery was excised, cannulated, and perfused with nominally Ca2⫹-free Tyrode solution containing 0.1% BSA for a period of ⬃5 min. Wedge preparations were then subjected to enzyme digestion with nominally Ca2⫹-free solution supplemented with 0.5 mg/ml collagenase (type II, Worthington) and 1 mg/ml BSA for 8 –12 min. After perfusion, thin slices of tissue from Epi (⬍2 mm from the Epi surface) and Endo (⬍2 mm from the Endo surface) were shaved from the wedge using a dermatome. Tissue slices were then placed in separate beakers, minced, incubated in fresh buffer containing 0.5 mg/ml collagenase and 1 mg/ml BSA, and agitated. The supernatant was filtered and centrifuged, and the pellet containing the myocytes was stored at room temperature. All animal procedures were in accordance with previously established guidelines (NIH Pub. No. 85-23, Revised 1985). Voltage-clamp recordings of peak INa. Early INa was measured as previously described (16) with minor modifications. Experiments were performed using a MultiClamp 700A (Axon Instruments, Foster AJP-Heart Circ Physiol • VOL

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City, CA). Command voltages were delivered, and data were acquired via a DigiData 1322 computer interface using Axon Instruments’ pCLAMP 9 program suite with data stored on a computer hard disk. Patch pipettes were pulled from borosilicate glass (1.5 mm outer diameter and 1.1 mm inner diameter) on a model PP-830 vertical puller (Narashige Instruments). The electrode resistance was 1–2.5 M⍀ when pipetters were filled with the internal solution (see below). The membrane was ruptured by applying negative pressure, and series resistance was compensated by 75– 80%. Whole cell current data were acquired at 20 –50 kHz and filtered at 5 kHz. Currents were normalized to cell capacitance. The external solution contained (in mM) 120 choline Cl, 10 NaCl, 2.8 Na⫹ acetate, 0.5 CaCl2, 4 KCl, 1 MgCl2, 1 CoCl2, 10 glucose, 10 HEPES, 0.1 BaCl2, and 5 NaOH; pH was adjusted to 7.4 with NaOH/HCl. The pipette solution contained (in mM) 15 NaCl, 145 Cs aspartate, 1 MgCl2, 5 KCl, 10 HEPES, 4 Na2ATP, and 10 EGTA; pH was adjusted to 7.2 with CsOH. Peak INa was dramatically reduced in the low extracellular Na⫹ to ensure adequate voltage control, as gauged by the slope of a Boltzmann fit to the steady-state activation curve (18). When Na⫹ channel kinetics and density were measured, the holding potential was ⫺120 mV to recruit all available Na⫹ channels. In addition, recordings of INa were made at least 5 min after rupture to minimize the effects of the time-dependent negative shift of

Fig. 1. Right ventricular (RV) wedge preparation exposed to increasing extracellular K⫹ concentrations ([K⫹]o). A: basic cycle length (BCL) of 2,000 ms. B: BCL of 300 ms. Endocardial (Endo; top) and epicardial (Epi; middle) action potentials (APs) and transmural ECGs are shown. Recordings were obtained under control conditions ([K⫹]o ⫽ 4 mM) and after perfusion of the preparations with 6, 8, 10, and 12 mM [K⫹]o. All AP recordings (obtained with floating microelectrodes) are scaled up to the amplitude of control recordings. Please note that the 2-to-1 Epi AP response at [K⫹]o ⫽ 12 mM shown in B (BCL ⫽ 300 ms) justifies the alternating T wave observed in the ECG. 295 • JULY 2008 •

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steady-state inactivation that occurs in conventional voltage-clamp experiments (36). Whole cell currents were analyzed using the Clampfit analysis program from pCLAMP 9 (Axon Instruments). Statistics. Results from pooled data are presented as means ⫾ SE. Statistical analysis was performed using ANOVA coupled with Tukey’s or Dunnett’s test (tissue slices and wedge data) or test followed by a Student-Newman-Keuls test or a Student t-test (cell data), as appropriate, using SigmaStat software. P ⬍ 0.05 was considered statistically significant. RESULTS

Figure 1 shows differences in the response of Endo and Epi APs and the resulting change in the transmural ECG caused by increasing [K⫹]o in an arterially perfused wedge preparation isolated from the RV. Endo and Epi APs and a transmural ECG were recorded under control conditions ([K⫹]o ⫽ 4 mM) and after perfusion of the preparation with Tyrode solution containing 6, 8, 10, and 12 mM [K⫹]o. All APs shown in Fig. 1 are scaled up to the amplitude of control signals (i.e., to Endo and Epi AP amplitudes recorded at [K⫹]o ⫽ 4 mM). Figure 1, A and B, displays signals recorded at BCLs of 2,000 and 300 ms, respectively. Figure 1 shows a preferential rate-dependent depression of Epi AP morphology following exposure to high [K⫹]o, particularly at concentrations of 10 and 12 mM and at the fastest stimulation rate (BCL ⫽ 300 ms). Thus, the differential response and slowing of transmural conduction underlie the R-ST segment changes resembling a MAP (MAP-like ECG), whereas the negative T wave is the result of a reversal of transmural repolarization gradients. At a BCL of 300 ms and [K⫹]o ⫽ 12 mM (Fig. 1B), transmural conduction failed during alternate beats leading to a 2:1 Epi AP response and subse-

quently conduction block and Epi unresponsiveness. Under these conditions, switching the pacing site from the Endo to Epi surface could not elicited a response, even at 10 times the control ET intensity. It needs to be recognized, however, that a reduced coupling in the sub-Epi (30, 35, 37), leading to a slow rate of charge transferred into this region, may in part be the cause of the greater AP changes observed in Epi, suggesting that the Endo versus Epi differential response to high [K⫹]o may be the consequence of an ensemble of effects. Conversely, since we observed a similar phenomenon in isolated Epi slices (see Fig. 4) as well as when we paced the wedge preparations from the Epi side (see Endo and Epi ETs in Fig. 3 and Tables 1–3), the referred reduced coupling is unlikely to affect the principal findings of this study. Figure 2 shows an experiment performed in a RV wedge preparation in which 12 mM [K⫹]o was perfused in the absence (A and B; BCLs ⫽ 2,000 and 300 ms, respectively) and presence of 1 mM 4-aminopyridine (4-AP; C and D). Figure 2 shows that, even in the presence of 4-AP, an Ito blocker, a preferential rate-dependent depression of Epi AP morphology following exposure to high [K⫹]o persisted. Thus, these results emphasize the notion that the differential response between Endo and Epi in AP morphology and excitability are unrelated to transmural differences in Ito. More readily, however, the data suggest that Endo versus Epi differences in INa underlie the excitability disparities. Similar results were found in two additional experiments performed in RV wedge preparations. The average data from the wedge experiments shown in Table 1 demonstrate that the QT interval and/or the AP duration at 50% and 90% repolarization of Endo and Epi

Fig. 2. Effect of 12 mM [K⫹]0 in the presence of 4-aminopyridine (4-AP). High [K⫹]o was perfused in the absence [BCLs ⫽ 2,000 (A) and 300 ms (B)] and presence of 1 mM 4-AP [BCLs ⫽ 2,000 (C) and 300 ms (D)]. The preferential rate-dependent depression of Epi AP morphology following exposure to high [K⫹]o persisted, indicating that the differential response between Endo and Epi is unrelated to transmural differences in transient outward K⫹ current. Rather, the data suggest that differences in fast Na⫹ current (INa) underlie the excitability disparities between the two tissues. All AP recordings (obtained with floating microelectrodes) are scaled up to the amplitude of control recordings.

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Table 1. Wedge preparation parameters 关K⫹兴o

BCL ⫽ 2,000 ms Endo ET, mA Epi ET, mA Epi APD50, ms Endo APD50, ms Epi APD90, ms Endo APD90, ms QT interval T wave amplitude, %R wave amplitude R wave width, ms BCL ⫽ 500 ms Endo ET, mA Epi ET, mA Epi APD50, ms Endo APD50, ms Epi APD90, ms Endo APD90, ms QT interval T wave amplitude, %R wave amplitude R wave width, ms BCL ⫽ 300 ms Endo ET, mA Epi ET, mA Epi APD50, ms Endo APD50, ms Epi APD90, ms Endo APD90, ms QT interval T wave amplitude, %R wave amplitude R wave width, ms

4 mM

6 mM

8 mM

10 mM

12 mM

0.4⫾0.1 0.5⫾0.1 159.5⫾4.6 184.6⫾4.5 193.1⫾3.2 233.4⫾4.6 257.5⫾8.5 20.3⫾2.5 28.7⫾2.3

0.6⫾0.2 0.5⫾0.1 152.3⫾4.2 178.3⫾5.1 177.9⫾3.2 218.2⫾4.5 236.0⫾6.4 26.3⫾2.6 31.7⫾1.6

0.5⫾0.1 0.5⫾0.1 152.2⫾7.4 183.4⫾8.0 174.5⫾7.7 212.1⫾10.5 223.6⫾7.3 30.1⫾3.5 29.3⫾1.7

0.5⫾0.1 0.7⫾0.1 134.9⫾6.4* 161.2⫾8.9 154.3⫾6.3* 189.3⫾9.2‡ 215.0⫾10.3* 23.2⫾5.1 34.9⫾4.3

0.6⫾0.1 1.9⫾0.5†‡ 117.6⫾5.9‡ 148.9⫾7.9* 137.9⫾4.9* 164.6⫾7.3‡ 217.4⫾11.4* 0.1⫾12.5 62.1⫾10.4‡

0.4⫾0.1 0.5⫾0.1 135.8⫾4.7 172.5⫾15.0 171.5⫾5.5 212.7⫾14.6 217.0⫾5.2 14.8⫾2.8 28.9⫾1.8

0.6⫾0.2 0.5⫾0.1 121.3⫾5.7 144.2⫾4.8 148.8⫾6.4 172.3⫾7.9 200.8⫾7.1 16.5⫾3.4 29.8⫾1.7

0.5⫾0.1 0.5⫾0.1 125.5⫾6.8 141.2⫾4.7 145.2⫾6.7* 164.1⫾5.6* 204.4⫾11.3 17.8⫾2.8 30.6⫾1.9

0.5⫾0.1 0.7⫾0.1 109.1⫾4.0† 133.7⫾6.5 126.7⫾5.4‡ 158.4⫾7.3* 186.0⫾10.0 3.8⫾7.0 39.7⫾4.3

0.8⫾0.2‡ 3.4⫾0.6†‡ 100.7⫾6.5‡ 126.4⫾6.2* 121.3⫾7.1‡ 134.4⫾10.6* 198.9⫾9.0 ⫺35.5⫾10.9‡ 66.8⫾10.3‡

0.4⫾0.1 0.5⫾0.1 110.4⫾4.8 126.2⫾4.5 144.7⫾6.6 163.2⫾4.3 185.3⫾5.1 6.2⫾4.1 28.3⫾2.0

0.6⫾0.0 0.5⫾0.1 102.5⫾4.5 111.9⫾1.9 125.9⫾5.5 146.2⫾2.5 172.0⫾6.4 15.0⫾5.2 29.0⫾2.5

0.5⫾0.1 0.5⫾0.1 95.8⫾4.4 114.8⫾4.5 119.0⫾5.8 143.2⫾5.0 169.1⫾6.2 3.7⫾4.8 28.8⫾2.4

0.6⫾0.1 1.0⫾0.2 89.8⫾5.6 102.8⫾4.1 107.8⫾5.5 124.7⫾5.0 159.7⫾9.0 ⫺23.2⫾9.3* 41.1⫾5.3

1.2⫾0.5* 3.4⫾0.7†‡ 83.8⫾6.6 101.3⫾6.3 104.2⫾4.8 121.4⫾5.2 185.1⫾10.0 ⫺45.2⫾7.5‡ 74.4⫾12.1‡

Values are means ⫾ SE; n ⫽ 12. Excitability threshold (ET) and action potential (AP) and ECG parameters were measured under control conditions 关extracellular K⫹ concentration (关K⫹兴o) ⫽ 4 mM兴 and at 6, 8, 10, and 12 mM 关K⫹兴o. BCL, basic cycle length; Endo, endocardium; Epi, epicardium; APD50 and APD90, AP duration at 50% and 90% repolarization, respectively. *P ⬍ 0.05 vs. 4 mM 关K⫹兴o; †P ⬍ 0.01 vs. Endo with 12 mM 关K⫹兴0; ‡P ⬍ 0.001 vs. 4 mM 关K⫹兴o.

significantly decreased with increasing concentrations of [K⫹]o. In addition, the ET significantly increased at 12 mM [K⫹]o (see also Fig. 3) relative to control (4 mM [K⫹]o) except in Endo at a BCL of 2,000 ms. Significant differences at 12 mM [K⫹]o were also found between Epi and Endo ET at all BCLs studied. Mean data of ET, R wave width, and T wave amplitude obtained from 12 wedge preparations (7 isolated from the RV and 5 isolated from the LV) are also graphically illustrated in Fig. 3. The differential response to high extracellular K⫹ was also observed between isolated Endo and Epi tissue slices in which the use of the standard microelectrode technique allowed us to accurately quantify all AP parameters, including AP amplitude, resting membrane potential (RMP), and Vmax since the impalements were stable from the beginning to end of each experiment measured. Figure 4A shows the response of isolated RV Endo (top) and Epi (bottom) to Tyrode solution containing 12 mM [K⫹]o at a BCL of 800 ms. Consecutive sets of Endo and Epi signals and correspondent Vmax values are depicted at 5-mV depolarization intervals starting at a RMP of ⫺86 mV. Note the marked depression of the response of Epi but not Endo when RMP reached a value of ⫺66 mV (5 consecutive Endo and Epi APs superimposed). It is noteworthy that the two tissues depolarized with a similar time course (Tables 2 and 3). The composite data shown in Fig. 4B demonstrate that at a RMP of ⫺66 mV, Vmax decreased to 51.1 ⫾ 5.3% of control in AJP-Heart Circ Physiol • VOL

Endo (n ⫽ 9) and 26.8 ⫾ 9.6% of control in Epi (n ⫽ 14, P ⬍ 0.05 vs. Endo). Table 2 also shows that the amplitude of phase 0 is greatly reduced in Epi as the membrane potential depolarizes to values positive to ⫺78 mV (P ⬍ 0.01 vs. Endo). The data obtained in wedge preparations and tissue slices show a greater sensitivity of Epi versus Endo to membrane depolarization, a condition that reduces Na⫹ channel availability. These observations suggest that there are differences in INa between the two tissues. To test this hypothesis, we measured early INa in Epi and Endo cells under voltage-clamp conditions. We first compared the current-voltage (I-V) relationship between Epi and Endo cells (Fig. 5). A set of 30-ms depolarizing pulses were applied in 5-mV increments from a holding potential of ⫺120 mV. The results of these experiments demonstrate that the density of INa was not different between Epi and Endo cells (Fig. 5C). We next tested the hypothesis that the difference in sensitivity between Epi and Endo to membrane depolarization is due to changes in steady-state gating parameters. Steady-state inactivation of INa was evaluated using a standard prepulse-test pulse voltage-clamp protocol (Fig. 6, top). The peak current following a 500-ms prepulse was normalized to the maximum current and plotted as a function of the prepulse voltage to obtain the availability of the channels. Data were fitted to a Boltzman function and yielded mid-inactivation voltages of ⫺83.6 ⫾ 0.1 mV for Epi and ⫺75.5 ⫾ 0.3 mV for Endo cells 295 • JULY 2008 •

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sentative traces in Epi and Endo cells. Reactivation of INa at ⫺100 mV for both cell types showed a fast and slow phase of recovery as follows: ␶1 ⫽ 14.5 ⫾ 0.8 ms and ␶2 ⫽ 65.7 ⫾ 9.7 ms for Epi cells and ␶1 ⫽ 12.8 ⫾ 0.6 ms and ␶2 ⫽ 45.6 ⫾ 2.5 ms for Endo cells (where ␶1 is the time constant for fast recovery and ␶2 is the time constant for slow recovery). The slow phase of recovery of INa was significantly slower in Epi cells (P ⬍ 0.05; Fig. 8C), suggesting that fewer Na⫹ channels are available at fast pacing frequencies. DISCUSSION

Fig. 3. Effect of increases in [K⫹]o on Endo excitability threshold (ET; top left) and Epi ET (top right) and on the width of the ECG R wave (bottom left) and T wave amplitude (expressed as a percentage of the R wave; bottom right). Values are means ⫾ SE; n ⫽ 12. *P ⬍ 0.05 vs. 4 mM [K⫹]o; £P ⬍ 0.01 vs. Endo with 12 mM [K⫹]o; ‡P ⬍ 0.001 vs. 4 mM [K⫹]o.

(P ⬍ 0.05; Fig. 6C). Thus, the availability of INa at normal RMP (approximately ⫺90 mV) is slightly greater in Endo than Epi. However, upon depolarization, these differences become accentuated, explaining why Epi cells tend to lose excitability earlier. Since Epi cells have a more negative steady-state inactivation voltage compared with Endo cells, it suggests that as cells depolarize, Epi cells would lose excitability at more negative voltages. To further test this hypothesis, we measured the magnitude of INa at different holding potentials. In the next set of experiments, the I-V relation was determined at holding potentials of ⫺120, ⫺90, and ⫺70 mV in the same cell. Data were normalized to the maximum current measured from a holding potential of ⫺120 mV. Figure 7 shows I-V relations recorded from Epi (A) and Endo cells (B) at different holding potentials. As the holding potential was more depolarized, there was a decrease in the size of INa. Figure 7C shows the amount of INa at different potentials. At ⫺70 mV, there was a significant reduction in INa in Epi cells compared with Endo cells. We next evaluated the time course of recovery from inactivation in the two cell types. Figure 8, A and B, shows repreAJP-Heart Circ Physiol • VOL

Although the differential sensitivity of ventricular Epi versus Endo to conditions encountered during AMI, hyperkalemia, and Brugada syndrome are well documented, (1, 9 –12, 15, 19, 25, 27, 31, 32, 39), the underlying basis for this distinctive behavior is not completely understood. Epi/Endo differences have, in the past, been largely attributed to differences in the contribution of Ito and/or IK,ATP (13, 22). The present study demonstrates another important electrophysiological distinction that contributes to the differential depression of Endo and Epi. Our data show differences in the inactivation characteristics of Na⫹ channels in the two cell types. Steady-state inactivation in Epi displays a half-inactivation voltage that is ⬃8 mV more negative and a recovery from inactivation that is slower than that of Endo. Our results strongly suggest that these distinctions contribute to a greater rate-dependent depression of Epi, particularly at depolarized potentials, leading to the development of transmural gradients of repolarization, disparate repolarization times, conduction slowing, and block, all of which contribute to arrhythmogenesis. Contrary to our findings, early INa differences in density but not in channel availability have been reported between Epi and Endo in the rat LV (3). The molecular basis for the differences in steady-state inactivation is not known. It is tempting to speculate that differences in the stochiometry of the Na⫹ channel’s auxiliary ␣and ␤-subunits underlie the lower Na⫹ channel availability observed in Epi. The more negative half-inactivation voltage observed in Epi may be secondary to a lower ␣-to-␤1-to-␤3 ratio, that is to say, a proportionally greater share of ␤-subunitforming proteins. An overall reduction in Na⫹ channel availability induced by the ␤-subunit has previously been reported (20). However, it needs to be recognized that coexpression of the Na⫹ channel’s ␣- and ␤-subunits has produced controversial electrophysiological results (28). Thus, the molecular basis for functionally distinct Na⫹ channels awaits further investigation. Clinical implications. Our data reveal that, in addition to IK,ATP and Ito, differences in early INa between Endo and Epi may contribute to the mechanisms involved in their differential response to AMI and other conditions that depolarize the ventricular myocardium (i.e., hyperkalemia). Although our measurements of inactivation characteristics were limited to LV myocytes, the qualitatively similar responses of RV and LV wedge preparations and tissue slices suggest that these distinctions in inactivation characteristics may apply as well to the RV. If so, these differences in Na⫹ channel characteristics between Epi and Endo may also contribute to the development of Brugada syndrome. 295 • JULY 2008 •

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Fig. 4. Progressive electrophysiological changes following superfusion with Tyrode solution containing 12 mM [K⫹]o (BCL⫽ 800 ms). A: Endo and Epi APs recorded from isolated RV tissue slices using the standard microelectrode technique. Vmax traces are shown underneath the respective APs. Consecutive sets of recordings were obtained at 5-mV depolarization intervals between the resting membrane potentials (RMPs) of ⫺86 and ⫺66 mV. Right traces show 5 consecutive beats of Endo and Epi APs superimposed. B: Vmax as a function of membrane depolarization in Endo and Epi. Values are means ⫾ SE; n ⫽ 9 Endo and 14 Epi. *P ⬍ 0.05 vs. RMP of ⫺86 mV.

however, that loss of function mutations in SCN5A may affect differentially Endo and Epi as long as they unmask, in a similar manner, the functional differences revealed at depolarized potentials reported in this study. Of note, many of the mutations associated with Brugada syndrome have been shown to alter the availability of INa by shifting the half-inactivation voltage to more negative potentials (33). Thus, we speculate that interventions designed to shift half-inactivation voltage to more positive potentials may be of therapeutic value. Finally, we need to recognize the fact that, as expected, the ECGs recorded from the wedge preparation may not fully replicate the in situ observations in that the surface ECG is an expression of a much more complex summation of voltage gradients including transmural (as in the wedge) and tangen-

It is well known that hyperkalemia is among the many clinical situations that may unmask or exacerbate the ECG pattern of Brugada syndrome (2). However, by no means are we proposing that our experiments with high [K⫹]o and the similarities between the wedge ECG and surface ECG in Brugada syndrome patients imply an involvement of depolarized tissues in the pathophysiology of this genetic disease. Rather, our data suggest that in the background of transmural and interventricular differences in Ito (11, 23, 38) and probably other conditions yet to be identified, the intrinsic functional differences in INa between Endo and Epi reported in this study may contribute to the manifestation of the disease, particularly in the 20 –30% of the cases in which a loss of function mutation in the SCN5A gene is found. It needs to be acknowledged, Table 2. Tissue slice parameters Vmax, mV/ms

RMP, mV ⫺86 ⫺84 ⫺82 ⫺80 ⫺78 ⫺76 ⫺74 ⫺72 ⫺70 ⫺68 ⫺66

Normalized Vmax, %

Endo

Epi

Endo

228.1⫾18.8 216.1⫾17.3 213.2⫾16.8 204.5⫾15.7 193.0⫾16.3‡ 185.0⫾15.1‡ 168.3⫾17.5§ 152.0⫾21.0§ 142.5⫾19.5§ 130.8⫾18.4§ 116.9⫾15.8§

188.6⫾15.5 180.1⫾14.0 170.5⫾13.2 159.2⫾12.3 146.8⫾11.2§ 133.3⫾10.7§ 111.9⫾10.3§ 91.6⫾10.3§ 76.5⫾10.4§ 68.2⫾9.1§ 39.8⫾11.6§

100.0⫾0.0 95.6⫾3.7 94.4⫾3.8 90.7⫾3.5 84.9⫾2.5* 81.8⫾2.9* 73.4⫾3.5§ 64.6⫾5.9§ 61.3⫾5.4§ 56.8⫾5.9§ 51.1⫾5.3§

Membrane Voltage at the End of Phase 0, mV

Epi

100.0⫾0.0 96.0⫾1.2 91.2⫾2.2 85.5⫾2.8§ 79.3⫾3.3§ 72.4⫾3.9§ 61.7⫾4.9§ 51.4⫾5.7§ 43.1⫾6.3§ 38.6⫾6.6§ 26.8⫾9.6†§

Recording Time, ms

Endo

Epi

Endo

Epi

22.5⫾3.6 24.8⫾2.5 24.8⫾2.7 24.3⫾2.8 24.2⫾2.9 23.1⫾3.0 22.0⫾3.2 20.9⫾3.3 19.8⫾3.4 18.1⫾3.3* 17.0⫾3.2*

12.3⫾1.6 12.1⫾1.5 12.1⫾1.5 11.5⫾1.4 10.4⫾1.5 9.5⫾1.4† 7.6⫾1.6† 6.1⫾1.6*† 3.7⫾2.7†§ 2.5⫾3.0†§ ⫺9.6⫾8.2†§

0.0⫾0.0 50.1⫾21.8 74.8⫾20.5 122.7⫾17.9 161.2⫾20.4 203.1⫾27.3 258.7⫾39.9 322.9⫾51.3 439.3⫾71.8 527.6⫾69.9 629.0⫾69.1

0.0⫾0.0 71.7⫾21.3 74.9⫾19.2 109.5⫾17.1 146.4⫾16.2 176.0⫾16.1 217.9⫾17.7 287.5⫾30.8 381.4⫾58.6 480.8⫾73.2 558.7⫾75.2

Values are means ⫾ SE; n ⫽ 9 Endo and 14 Epi. Vmax, change in voltage over time of phase 0; normalized Vmax, Vmax normalized to a resting membrane potential (RMP) of ⫺86 mV; recording time, time taken for Endo and Epi APs to depolarize, in 2-mV intervals, from a RMP of ⫺86 mV (time 0). *P ⬍ 0.05 vs. RMP of ⫺86 mV; †P ⬍ 0.05 vs. Endo; ‡P ⬍ 0.01 vs. RMP of ⫺86 mV; §P ⬍ 0.001 vs. RMP of ⫺86 mV. AJP-Heart Circ Physiol • VOL

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Table 3. ETs of Endo and Epi tissue slices

Endo Epi

4 mM 关K⫹兴

12 mM 关K⫹兴

0.37⫾0.04 0.50⫾0.07

0.44⫾0.05 ⱖ5.0

Values are means ⫾ SE (in mA); n ⫽ 9 Endo and 14 Epi.

tial, the latter comprising apicobasal, interregional, and interventricular gradients. Summary. Our study demonstrates important differences in early INa between canine ventricular Endo and Epi. The data suggest that reduced INa availability in Epi may contribute to its greater sensitivity to electrical depression and may in part contribute to electrocardiographic changes and arrhythmogenesis under a wide variety of pathophysiological conditions. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Judy Hefferon and Dr. Arthur Iodice. GRANTS This work was supported by grants from the American Health Assistance Foundation (to J. M. Cordeiro), National Heart, Lung, and Blood Institute Grant HL-47678 (to C. Antzelevitch), and a grant from the American Heart Association (to J. M. Di Diego). REFERENCES 1. Antzelevitch C. The Brugada syndrome: ionic basis and arrhythmia mechanisms. J Cardiovasc Electrophysiol 12: 268 –272, 2001. 2. Antzelevitch C, Brugada P, Borggrefe M, Brugada J, Brugada R, Corrado D, Gussak I, LeMarec H, Nademanee K, Perez Riera AR, Shimizu W, Schulze-Bahr E, Tan H, Wilde A. Brugada syndrome: report of the second consensus conference. Heart Rhythm 2: 429 – 440, 2005.

Fig. 6. Representative steady-state inactivation recordings for Epi (A) and Endo (B) observed in response to the voltage-clamp protocol (top). C: steadystate inactivation relation for the two cell types. Peak currents were normalized to their respective maximum values and plotted against the conditioning potential. Epi cells showed a midinactivation potential that was significantly hyperpolarized compared with Endo cells.

Fig. 5. Representative whole cell current recordings from an Epi (A) and Endo (B) left ventricular (LV) myocyte. Current recordings were obtained at test potentials between ⫺80 and 10 mV in 5-mV increments from a holding potential (HP) of ⫺120 mV. C: current-voltage (I-V) relations for Epi (n ⫽ 14) and Endo (n ⫽ 15) cells showing no differences in current density. D: steady-state activation relations for Epi and Endo. Chord conductance was determined using the ratio of current to the electromotive potential for the cells shown in C. Data were normalized and plotted against their test potential. AJP-Heart Circ Physiol • VOL

3. Ashamalla SM, Navarro D, Ward CA. Gradient of sodium current across the left ventricular wall of adult rat hearts. J Physiol 536: 439 – 443, 2001. 4. Billman GE. Ro 40 –5967, a novel calcium channel antagonist, protects against ventricular fibrillation. Eur J Pharmacol 229: 179 –187, 1992. 5. Chen Q, Kirsch GE, Zhang D, Brugada R, Brugada J, Brugada P, Potenza D, Moya A, Borggrefe M, Breithardt G, Ortiz-Lopez R, Wang Z, Antzelevitch C, O’Brien RE, Schultze-Bahr E, Keating MT, Towbin JA, Wang Q. Genetic basis and molecular mechanisms for idiopathic ventricular fibrillation. Nature 392: 293–296, 1998. 6. Cordeiro JM, Greene L, Heilmann C, Antzelevitch D, Antzelevitch C. Transmural heterogeneity of calcium activity and mechanical function in the canine left ventricle. Am J Physiol Heart Circ Physiol 286: H1471– H1479, 2004. 7. Di Diego JM, Antzelevitch C. Pinacidil-induced electrical heterogeneity and extrasystolic activity in canine ventricular tissues. Does activation of ATP-regulated potassium current promote phase 2 reentry? Circulation 88: 1177–1189, 1993. 8. Di Diego JM, Antzelevitch C. High [Ca2⫹]-induced electrical heterogeneity and extrasystolic activity in isolated canine ventricular epicardium. Phase 2 reentry. Circulation 89: 1839 –1850, 1994. 295 • JULY 2008 •

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Fig. 7. I-V relations for Epi (A) and Endo (B) cells showing a reduction in current as HP is more depolarized. n ⫽ 8 cells from Epi and Endo. C: bar graph showing the maximum current available at different HPs from the two cell types. *P ⬍ 0.05.

Fig. 8. Representative traces recorded from an Epi (A) and Endo cell (B) showing the recovery of INa. Recovery was measured using two identical voltage-clamp steps to ⫺20 mV from a HP of ⫺100 mV separated by selected time intervals. The recovery time course of INa, recorded from the two cell types (C), was fit by two exponentials. The slow phase of recovery was significantly slower in Epi cells (P ⬍ 0.05; C).


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9. Di Diego JM, Antzelevitch C. Cellular basis for ST-segment changes observed during ischemia. J Electrocardiol 36, Suppl: 1–5, 2003. 10. Di Diego JM, Cordeiro JM, Goodrow RJ, Fish JM, Zygmunt AC, Perez GJ, Scornik FS, Antzelevitch C. Ionic and cellular basis for the predominance of the Brugada syndrome phenotype in males. Circulation 106: 2004 –2011, 2002. 11. Di Diego JM, Sun ZQ, Antzelevitch C. Ito and action potential notch are smaller in left vs. right canine ventricular epicardium. Am J Physiol Heart Circ Physiol 271: H548 –H561, 1996. 12. Elharrar V, Gaum WE, Zipes DP. Effects of drugs on conduction delay and the incidence of ventricular arrhythmias induced by acute coronary occlusion in dogs. Am J Cardiol 39: 544 –549, 1977. 13. Furukawa T, Kimura S, Furukawa N, Bassett AL, Myerburg RJ. Role of cardiac ATP-regulated potassium channels in differential responses of endocardial and epicardial cells to ischemia. Circ Res 68: 1693–1702, 1991. 14. Gasparini M, Priori SG, Mantica M, Napolitano C, Galimberti P, Ceriotti C, Simonini S. Flecainide test in Brugada syndrome: a reproducible but risky tool. Pacing Clin Electrophysiol 26: 338 –341, 2003. 15. Gilmour RF Jr, Zipes DP. Different electrophysiological responses of canine endocardium and epicardium to combined hyperkalemia, hypoxia, and acidosis. Circ Res 46: 814 – 825, 1980. 16. Haufe V, Cordeiro JM, Zimmer T, Wu YS, Schiccitano S, Benndorf K, Dumaine R. Contribution of neuronal sodium channels to the cardiac fast sodium current INa is greater in dog heart Purkinje fibers than in ventricles. Cardiovasc Res 65: 117–127, 2005. 17. Hsieh MH, Cheng CY, Chan P, Tai CT, Chen SA. Monophasic action potential-like electrocardiogram simulating acute myocardial infarction. J Interv Card Electrophysiol 8: 41– 44, 2003. 18. Kaab S, Nuss HB, Chiamvimonvat N, O’Rourke B, Pak PH, Kass DA, Marban E, Tomaselli GF. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ Res 78: 262–273, 1996. 19. Kimura S, Bassett AL, Kohya T, Kozlovskis PL, Myerburg RJ. Simultaneous recording of action potentials from endocardium and epicardium during ischemia in the isolated cat ventricle: relation of temporal electrophysiologic heterogeneities to arrhythmias. Circulation 74: 401– 409, 1986. 20. Ko SH, Lenkowski PW, Lee HC, Mounsey JP, Patel MK. Modulation of Nav1.5 by beta1- and beta3-subunit co-expression in mammalian cells. Pflu¨gers Arch 449: 403– 412, 2005. 21. Krishnan SC, Antzelevitch C. Sodium channel block produces opposite electrophysiological effects in canine ventricular epicardium and endocardium. Circ Res 69: 277–291, 1991. 22. Li RA, Leppo M, Miki T, Seino S, Marban E. Molecular basis of electrocardiographic ST-segment elevation. Circ Res 87: 837– 839, 2000. 23. Litovsky SH, Antzelevitch C. Transient outward current prominent in canine ventricular epicardium but not endocardium. Circ Res 62: 116 – 126, 1988.


24. Littmann L, Monroe MH, Kerns WP, Svenson RH, Gallagher JJ. Brugada syndrome and “Brugada sign:” clinical spectrum with a guide for the clinician. Am Heart J 145: 768 –778, 2003. 25. Liu DW, Gintant GA, Antzelevitch C. Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle. Circ Res 72: 671– 687, 1993. 26. Lukas A, Antzelevitch C. Differences in the electrophysiological response of canine ventricular epicardium and endocardium to ischemia: role of the transient outward current. Circulation 88: 2903–2915, 1993. 27. Lukas A, Antzelevitch C. Phase 2 reentry as a mechanism of initiation of circus movement reentry in canine epicardium exposed to simulated ischemia. Cardiovasc Res 32: 593– 603, 1996. 28. Meadows LS, Isom LL. Sodium channels as macromolecular complexes: implications for inherited arrhythmia syndromes. Cardiovasc Res 67: 448 – 458, 2005. 29. Pastor JA, Castellanos A, Moleiro F, Myerburg RJ. Patterns of acute inferior wall myocardial infarction caused by hyperkalemia. J Electrocardiol 34: 53–58, 2001. 30. Poelzing S, Akar FG, Baron E, Rosenbaum DS. Heterogeneous connexin43 expression produces electrophysiological heterogeneities across ventricular wall. Am J Physiol Heart Circ Physiol 286: H2001–H2009, 2004. 31. Ruffy R, Lovelace ED, Knoebel SB, Mueller TM, Zipes DP. Relationship between changes in left ventricular bipolar electrograms and regional myocardial blood flow during acute coronary occlusion in the dog. Circ Res 45: 764 –770, 1979. 32. Scherlag BJ, El-Sherif N, Hope RR, Lazzara R. Characterization and localization of ventricular arrhythmias resulting from myocardial ischemia and infarction. Circ Res 35: 372–383, 1974. 33. Tan HL. Sodium channel variants in heart disease: expanding horizons. J Cardiovasc Electrophysiol 17, Suppl 1: S151–S157, 2006. 34. Vereckei A. Inferior wall pseudoinfarction pattern due to hyperkalemia. Pacing Clin Electrophysiol 26: 2181–2184, 2003. 35. Vetter FJ, Simons SB, Mironov S, Hyatt CJ, Pertsov AM. Epicardial fiber organization in swine right ventricle and its impact on propagation. Circ Res 96: 244 –251, 2005. 36. Wendt DJ, Starmer CF, Grant AO. Na channel kinetics remain stable during perforated-patch recordings. Am J Physiol Cell Physiol 263: C1234 –C1240, 1992. 37. Yamada KA, Kanter EM, Green KG, Saffitz JE. Transmural distribution of connexins in rodent hearts. J Cardiovasc Electrophysiol 15: 710 –715, 2004. 38. Yan GX, Antzelevitch C. Cellular basis for idiopathic VT/VF syndrome. Circulation 94: I-625, 1996. 39. Yan GX, Antzelevitch C. Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST segment elevation. Circulation 100: 1660 –1666, 1999.

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