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(Circulation Research. 1997;81:110-119.)
© 1997 American Heart Association, Inc.

Articles

Alterations of Na⁺ Currents in Myocytes From Epicardial Border Zone of the Infarcted Heart

A Possible Ionic Mechanism for Reduced Excitability and Postrepolarization Refractoriness

Jielin Pu, , Penelope A. Boyden

From the Department of Pharmacology, Columbia University, New York, NY.

Correspondence to Penelope A. Boyden, Department of Pharmacology, Columbia College of Physicians and Surgeons, 630 West 168th St, New York NY 10032. E-mail pab4{at}columbia.edu

	Abstract

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Abstract Previously, we have shown abnormalities in _max and in the recovery of _max in myocytes dispersed from the epicardial border zone (EBZ) of the 5-day infarcted canine heart (myocytes from the EBZ [IZs]). Thus, we sought to determine the characteristics of the whole-cell Na⁺ current (I_Na) in IZs and compare them with the I_Na of cells from noninfarcted hearts (myocytes from noninfarcted epicardium [NZs]). I_Na was recorded using patch-clamp techniques under conditions that eliminated contaminating currents and controlled I_Na for measurement (19°C, 5 mmol/L [Na⁺]_o). Peak I_Na density (at -25 mV) was significantly reduced in IZs (4.9±0.44 pA/pF, n=36) versus NZs (12.8±0.55 pA/pF, n=54; P<.001), yet the half-maximal activation voltage (V_0.5), time course of decay, and time to peak I_Na were no different. However, in IZs, V_0.5 of the availability curve (I/I_max curve) was shifted significantly in the hyperpolarizing direction (-80.2±0.48 mV in NZs [n=45] versus -83.9±0.59 mV in IZs [n=27], P<.01). Inactivation of I_Na directly from a depolarized prepotential (-60 mV) was significantly accelerated in IZs versus NZs (fast and slow time constants [₁ and ₂, respectively] were as follows: NZs [n=28], ₁=71.5±5.6 ms and ₂=243.7±17.1 ms; IZs [n=21], ₁=36.3±2.4 ms and ₂=153±11.3 ms; P<.001). Recovery of I_Na from inactivation was dependent on the holding potential (V_H) in both cell types but was significantly slower in IZs. At V_H=-90 mV, I_Na recovery had a lag in 18 (82%) of 22 IZs (with a 17.6±1.5-ms lag) versus 2 (9%) of 22 NZs (with 5.9- and 8.7-ms lags); at V_H=-100 mV, ₁=60.9±2.6 ms and ₂=352.8±28.1 ms in NZs (n=41) versus ₁=76.3±4.8 ms and ₂=464.4±47.2 ms in IZs (n=26) (P<.002 and P<.03, respectively); at V_H=-110 mV, ₁=33.4±1.8 ms and ₂=293.5±33.6 ms in NZs (n=21) versus ₁=44.3±2.9 ms and ₂=388.4±38 ms in IZs (n=18) (P<.002 and P<.07, respectively). In sum, I_Na is reduced, and its kinetics are altered in IZs. These changes may underlie the altered excitability and postrepolarization refractoriness of the ventricular fibers of the EBZ, thus contributing to reentrant arrhythmias in the infarcted heart.

Key Words: Na⁺ current • ion channel • ventricular myocyte • myocardial infarction • epicardial border zone

	Introduction

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Like cardiac ischemia, which leads to myocardial infarction, metabolic inhibition induced by hypoxia or pharmacological agents is accompanied by alteration in transmembrane action potentials of cardiac cells.^{1 2 3 4} However, the effects of metabolic inhibitors on the fast I_Na remain controversial.⁵ In voltage-clamped frog atrial trabeculae, metabolic inhibitors suppress I_Na⁶ or have no effect.⁷ Hypoxia in the presence of normal extracellular K⁺ solutions has only a slightly depressant effect on the maximal upstroke velocity, _max, and its recovery kinetics,^{4 8} whereas in the presence of high extracellular K⁺ solutions, hypoxia decreases _max and markedly prolongs the recovery kinetics of _max. These acute changes in Na⁺ channel function may underlie abnormalities in cardiac impulse conduction and refractoriness, leading to acute ischemic arrhythmias.

Na⁺ channel function may also be altered in cells that survive in the EBZ of the healing infarcted heart, since several studies have shown that _max values are reduced in fibers of the multicellular EBZ preparation of the 5-day infarcted heart.^{9 10 11 12} In addition, prolonged refractoriness and slowed conduction are thought to contribute to unidirectional block in the EBZ.^{9 13 14 15 16} Importantly, these fibers provide the substrate for serious reentrant ventricular tachycardias that have been shown to occur at this time after total coronary artery occlusion.¹⁷ In some studies, cells in the multicellular EBZ preparations had normal resting membrane potentials but had action potentials with reduced total potential amplitude and no overshoot.¹⁸ Furthermore, transmembrane potential alterations observed in the multicellular preparation are conserved at the single myocyte level, whereas reduced _max, a delay in recovery of _max, and a shift in the steady state availability of _max have been described for myocytes dispersed from the infarcted heart.^{11 12} It is entirely possible that abnormalities in Na⁺ channel function in myocytes from the EBZ may result from changes that first occurred during the acute ischemic phase and then persisted throughout this healing phase. However, to our knowledge, no one has reported on the function of I_Na in myocytes that have survived in the infarcted heart.

Therefore, we studied I_Na and its kinetics by means of the patch-clamp technique in the whole-cell recording configuration. We found that the I_Na density and kinetics are significantly altered in myocytes that survive in the EBZ of the infarcted heart.

	Materials and Methods

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Cell Preparation
Adult mongrel male dogs (12 to 18 kg, 1 to 2 years old) were used in the present study. Myocardial infarction was produced according to the Harris procedure.¹⁹ Under isoflurane anesthesia and sterile conditions, the LAD was isolated and completely occluded in two stages. If multiple ventricular beats occurred at the time of the surgical procedure, dogs were treated with lidocaine (2 mg/kg IV). After 5 to 6 days, a cardiectomy was performed while the dogs were anesthetized with sodium pentobarbital (30 mg/kg IV). The infarcted region of the heart was identified on gross examination as a pale white mottled area on the epicardial surface. A scalpel blade was used to quickly remove a thin slice (1 cmx2 cmx2 to 3 mm) of epicardial muscle (EBZ) from this area on the anterior left ventricle adjacent to the LAD.

Single Ca²⁺-tolerant ventricular myocytes were enzymatically dispersed from the slice of EBZ tissue (IZs) and from slices of epicardial tissue obtained in a similar fashion from the same anatomic region of control noninfarcted hearts (NZs), as previously described.¹¹ Briefly, the tissue was rinsed twice in a Ca²⁺-free solution that contained (mmol/L) NaCl 115, KCl 5, sucrose 35, dextrose 10, HEPES 10, and taurine 4, pH 6.95, to remove blood. Then it was triturated in 20 mL of enzyme-containing solution (0.44 mg/mL collagenase B, Boehringer-Mannheim Corp; 0.25 mg/mL protease, Sigma Chemical Co; 36°C to 37°C) for 30 minutes; after which, the solution was decanted and discarded. For a second trituration, the solution contained only collagenase and was discarded after 30 minutes. The next six to seven triturations were each performed for 15 minutes. Each time, the solution was centrifuged at 500 rpm for 3 minutes to collect the supernatant and dispersed myocytes. Resuspension solution was changed every 30 minutes for solutions containing increasing concentrations of Ca²⁺ (50 to 500 µmol/L). With this procedure, the living NZ yield was 30% to 40%. For NZs, only rod-shaped cells with staircase ends, clear cross striations, and surface membranes free from blebs were used for study. IZs chosen for study, however, had a ruffled appearance, were less rodlike appearing, and had somewhat irregular cross striations and small dark droplets on the membrane. Our recent studies show that these morphological features indicate the IZs exhibiting abnormal transmembrane action potentials that are similar to those of the multicellular preparation of the EBZ.^{9 10 11 20}

Experimental Conditions
For study, an aliquot of cells was transferred onto a poly-L-lysine–coated glass coverslip placed at the bottom of a 0.5-mL tissue chamber, which had been mounted on the stage of a Nikon inverted microscope (Nikon Diaphot). Myocytes were continuously superfused (2 to 3 mL/min) with normal Tyrode's solution containing (mmol/L) NaCl 137, NaHCO₃ 24, NaH₂PO₄ 1.8, MgCl₂ 0.5, CaCl₂ 2.0, KCl 4.0, and dextrose 5.5 (pH 7.4). The solution was bubbled with 5% CO₂/95% O₂. Temperature was continuously monitored and maintained at 19.0±0.5°C for proper voltage control. Patch pipettes were made from borosilicate thin wall glass (Sutter Instrument Co; outer diameter, 1.5 mm; inner diameter, 1.10 mm) using a Flaming/Brown-type horizontal puller (model P-87, Sutter Instrument Co). Each pipette tip was polished using a microforge (type MF-83, Narishige, Scientific Instrument Laboratories) just before use. Pipette resistances ranged between 0.6 to 0.9 M when filled with an internal solution that had the following composition (mmol/L): CsOH 125, aspartic acid 125, tetraethylammonium chloride 20, HEPES 10, Mg-ATP 5, EGTA 10, and phosphocreatine 3.6 (pH 7.3 with CsOH). After the formation of the gigaohm seal, the stray capacitance was electronically nulled. The cell membrane under the pipette tip was then ruptured by an brief increase in suction, forming the whole-cell recording configuration. A period of 7 minutes was then allowed for intracellular dialysis to begin before switching to the low-Na⁺ extracellular solution (mmol/L): NaCl 5, MgCl₂ 1.2, CaCl₂ 1.8, CsCl 5, tetraethylammonium chloride 125, HEPES 20, glucose 11, 4-aminopyridine 3, and MnCl₂ 2 (pH 7.3 with CsOH), designed for proper I_Na measurements. Whereas Mn²⁺ is known to affect I_Na,²¹ preliminary experiments showed that the effect on I_Na in IZs (n=4, 39.6% decrease) was similar to its effect in NZs (n=3, 39.1% decrease). With this combination of external and internal solutions, I_Na would be of manageable size and isolated from other possible contaminating currents. Time-dependent changes of Na⁺ channel kinetics, including a shift of the I/I_max curve in the hyperpolarizing direction have been observed.^{22 23 24} Typically, these changes are known to occur in the first 15 minutes after membrane rupture. In our experiments, therefore, 15 minutes was allowed to approach a stabilization in low-Na⁺ solution.

Voltage-Clamp and Recording Techniques
Whole-cell I_Na was recorded using the whole-cell patch-clamp technique. Voltage-clamp experiments were performed with an Axopatch 200A clamp amplifier (headstage, CV 201A; gain, ß=1; Axon Instruments). Clamp protocols were generated with a 16-bit digital-to-analog converter (Digidata 1200, Axon Instruments) controlled by PCLAMP software and a Gateway 2000 computer. The currents were filtered at 10 kHz, digitized at a sampling interval 0.1 ms for whole-cell currents and 0.02 ms for capacitative transients, and stored on the computer for later analysis. The membrane capacity (in pF) of each cell was measured in the Cs⁺-rich solution by integrating the area under a capacitative transient induced by a 10-mV hyperpolarizing clamp step (from -80 to -90 mV) and dividing this area by the voltage step. Current amplitude data of each cell were then normalized to its cell capacitance (current density [pA/pF]). Averaged cell capacitances were 133.4±6 pF in NZs (n=54) and 159.8±9 pF in IZs (n=36) (P<.05). The average time constant of decay of the capacitive transient was 0.159±0.03 ms in NZs and 0.163±0.03 ms in IZs (P>.05). Therefore, the residual series resistance for each cell was calculated to be 1.26±0.04 M in NZs and 1.08±0.1 M in IZs. Thus, average steady state voltage error resulting from series resistance was 2.0±0.1 mV for NZs and 0.75±0.1 mV for IZs (P>.01).

For consideration of the voltage control, we lowered extracellular Na⁺ concentration to 5 mmol/L, maintained the temperature at 19±0.5°C, and used patch pipettes only with resistances <1 M. Furthermore, we did not choose large cells. If experiments demonstrated evidence of inadequate voltage control, eg, a "threshold phenomenon" near the voltage range for Na⁺ channel activation, and/or an inappropriately steep increase in current amplitude in the negative slope region of the I-V relationship curve, data were discarded. For the other clamp protocols, there should not be a crossover of currents as their size changes, and the peak should occur at about the same time.^{25 26} Furthermore, in several experiments in cells from both groups, we recorded tail currents at time of peak currents to ascertain whether under our recording conditions we had adequate voltage control (data not shown). We also considered a criterion described by Hanck and Sheets²⁴ that the slope factor of activation curve should not be <-6 mV. Whole-cell I_Na was obtained by subtracting the traces elicited with comparable voltage steps containing no current (using prepulse or manipulating the V_H to inactivate the Na⁺ channels) from the raw current traces. In this way, the cell capacitance and linear leakage were subtracted.

Experimental Protocols
To examine the peak current density in cells from the two groups, voltage steps (50-ms duration) from a V_H of -100 mV were given stepwise from -70 to +5 mV. Peak current at various levels of V_t was plotted to obtain the I-V curve. The peak current at each V_t was then normalized to cell capacitance (pA/pF) to obtain a current density-voltage relationship curve. From I-V data, the potential dependence of activation of I_Na was derived by using the following equation:

where I_Na is the peak current at the test voltage (V_t), g_Na is the chord conductance, and E_rev is the reversal potential obtained from I-V curve, where the I-V curve crossed over the zero line. The g_Na versus voltage relationships were then fit to

to obtain V_0.5 and the slope factor k.

The time course of I_Na decay during a depolarizing pulse (development of inactivation) was best described by fitting the current trace between the inward peak and the end of the pulse using CLAMPFIT (CLAMP 5.0 software). The best fit was accepted if additional exponential components would either reduce the fit quality or prevent convergence altogether. "Steady state" availability (I/I_max) was characterized by using a double-pulse protocol. Here, a 1000-ms conditioning pulse to various potentials (from -120 to -50 mV ) was followed by a 2-ms interval back to V_H (-100 mV) before a 40-ms test pulse to -25 mV. Each double-pulse protocol was separated by a 5-s recovery interval. From these data, the steady state inactivation curve for each cell was obtained by normalizing currents to the maximal current elicited from a conditioning potential of -120 mV. The Boltzmann equation was used to describe data and to obtain V_0.5 and the slope factor k.

In a subset of cells, the time course of inactivation directly from the "closed state" was determined by a double-pulse method.²⁷ A prepulse from -100 to -60 mV of variable duration (from 1 to 1000 ms) was used. This pulse was a subthreshold pulse and did not elicit I_Na. After the prepulse, the cell membrane was clamped back to -100 mV for a 2-ms interval, followed by test pulse to V_t=-25 mV. The current elicited by a test pulse after each prepulse duration was normalized to the maximal current obtained when the prepulse duration equaled 1 ms. By plotting the normalized current against the duration of the prepulse, we described the decrease in the test pulse I_Na by the sum of two exponentials^{27 28} :

where Amp1 and Amp2 are the relative amplitudes of ₁ and ₂, respectively.

The time course of recovery of I_Na from steady state inactivation was assessed at three repolarized potentials using the double-pulse method. Here, a 1000-ms conditioning pulse (V_H, -100 to -25 mV) was followed by a variable IPI at a potential of -90, -100, or -110 mV, and then a test pulse (to -25 mV) was given to elicit the I_Na. Between each set of pulses, a 5-s resting interval was introduced. I_Na elicited by each test pulse was then normalized to the maximal current value obtained at IPI=3000 ms. A biexponential function^{26 27 29} was used to describe the change in normalized values plotted against IPI. In some cells, an estimate of the magnitude of the initial delay was set as a lag in recovery, which was determined as the longest IPI that elicited no I_Na. This was compared with the fit delay (d), which was obtained by using the following equation:

In this way, the time constant of recovery from steady state inactivation was determined for each NZ and IZ, and the average values were compared.

Statistics
All values are represented as mean±SEM. A value of P<.05 was considered statistically significant. For a two-sample comparison, an unpaired t test was used to compare a single mean value between the two independent cell groups. A paired t test was used to compare the mean values obtained from the same cell group before and after an intervention (eg, in comparing V_0.5 values obtained with a different duration of prepulse in the same cell group). For multiple comparisons, such as to compare V_H-dependent recovery time course of I_Na in each group, an ANOVA was used, followed by an F test to determine if the sample mean values between groups were significantly different from each other. If so, a modified t test with Bonferroni's correction was used (Sigmastat, Jandel Scientific).

	Results

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To minimize the difference between I_Na amplitudes in NZs and IZs that were secondary to shifts in the inactivation curve and to decide on a time frame in which the data collected were comparable in both groups, we first studied the rate of I_Na amplitude changes and changes in the V_0.5 value of I/I_max curves with time after membrane rupture. From 20 to 50 minutes after membrane rupture, V_0.5 values showed a similar rate of shifting, 0.14±0.037 mV/min in NZs (n=7) and 0.11±0.029 mV/min in IZs (n=7) (P>.05). Therefore, peak current data collected between 23 and 50 minutes after membrane rupture were used. In the analysis of data from individual protocols, we compared the average time after membrane rupture at which data were collected for cells in order to correctly make comparisons between groups.

I_Na Density and Activation
We determined peak current density-voltage relationships of I_Na for enzymatically isolated ventricular epicardial border zone cells. Fig 1 shows a family of capacitance and leak-subtracted I_Na traces obtained in a typical NZ (Fig 1A) and IZ (Fig 1B). The average amplitude of peak I_Na was reduced more significantly in IZs (-758.5±75.8 pA, n=36) than in NZs (-1722.2±111.8 pA, n=54; P<.001). The current measured in cells of both groups was I_Na, because it was totally blocked by 10 µmol/L tetrodotoxin (data not shown). Fig 2A illustrates the average peak density-voltage relationship curve of I_Na for 54 NZs and 36 IZs. For both cell types, the current activation occurred at -55 mV. With further depolarization, currents gradually increased in magnitude, reaching peak density at V_t=-25 mV and reversing near 0 mV. This is similar to the findings of others.^{27 30 31} I_Na density was reduced significantly in IZs at V_t=-45 to -5 mV compared with that of NZs. The peak I_Na density was 12.8±0.6 pA/pF in NZs and only 4.9±0.4 pA/pF in IZs (P<.001), representing a 61.7% decrease. Data used to construct these density-voltage relationships were obtained at similar times after membrane rupture (23.6±0.33 minutes in NZs and 23.2±0.4 minutes in IZs). When average density-voltage curves were normalized by setting the peak density to 1 in each group, the normalized current density-voltage curves from NZs and IZs were superimposable (Fig 2B).

View larger version (23K): [in this window] [in a new window]   Figure 1. Family of tracings of INa obtained for I-V relationships. INas were elicited from a VH of -100 mV to various levels of Vt (-70 to +5 mV) for a 50-ms duration in an NZ (141 pF, A) and IZ (150 pF, B). The calibration bar is for both panels. Membrane capacity and linear leakage were subtracted for each current tracing (see "Materials and Methods").

View larger version (22K): [in this window] [in a new window]   Figure 2. Averaged INa density-voltage relationships in NZs and IZs. A, Mean±SEM INa density at each Vt is plotted as a function of Vt (VH=-100 mV). The data were obtained 23.6±0.33 minutes in NZs and 23.3±0.4 minutes in IZs after cell membrane rupture. Average cell capacity was 133.4±6 pF in NZs and 159.8±9 pF in IZs. indicates averaged values for NZs; , averaged values for IZs. *P<.05 vs NZs at Vt. B, Averaged INa density-voltage relationships of panel A were normalized by taking the maximal current density as 1 in each group. Normalized density-voltage relationship curves show a similar shape for NZs and IZs.

The average peak density of I_Na differed between the two groups, and so did the distribution of I_Na within a group (Fig 3). Note that in NZs, I_Na ranged from 23 to 6 pA/pF, with most cells in the 10- to 15-pA/pF range (69%). For IZs, some cells fell into the low range of NZ values (5 to 10 pA/pF), but a majority of IZs had I_Na of <5 pA/pF (58.3%).

View larger version (34K): [in this window] [in a new window]   Figure 3. Distribution of INa density. The number of cells having the same current density is plotted against the magnitude of current density for NZs (open bars) and IZs (hatched bars). The height of the bar indicates the total number of cells with INa density. INa density in most NZs was in the range of 10 to 15 pA/pF (69%); most IZs (58.3%) had INa ranging from 3 to 5 pA/pF.

Time Course of I_Na Peak and Decay
The time course of I_Na decay during maintained depolarization was voltage dependent. A monoexponential function was used to fit current decay at all V_t values in cells of both groups (Table 1). With a longer test pulse (100 ms), the decay of I_Na was fit to a biexponential function; however, the slower time constant existed only at V_t of -40 mV (data not shown). Furthermore, the contribution of the slower component to the total current decay was 10%. Therefore, the time constants responsible for the fast component of current decay were evaluated and used for comparison. There were no differences between NZs and IZs (Table 1).

View this table: [in this window] [in a new window]   Table 1. INa During Maintained Depolarization

In all cells used for peak I_Na measurements, time to peak of current was measured. There were no differences between NZs and IZs (Table 1).

Activation and Inactivation of I_Na
We used a double-pulse protocol to determine typical steady state inactivation (I/I_max) curves. Fig 4 shows selected current traces recorded from a typical NZ (Fig 4A) and IZ (Fig 4B). In Fig 4C, average normalized I_Na values for each cell are plotted as a function of V_c. Data plotted were collected at 25.2±0.5 minutes in NZs and 25.9±0.7 minutes in IZs after rupture of the cell membrane (P>.05). The average maximally available I_Na (at V_c=-120 mV) was 12.0±0.7 pA/pF in NZs and 5.2±0.5 pA/pF in IZs (P<.001). In IZs, V_0.5 of the I/I_max curve was significantly shifted in the hyperpolarizing direction (-80.2±0.5 mV in NZs [n=45] versus -83.9±0.6 mV in IZs [n=27], P<.01). The slope factors were no different (P>.05). g_Na was plotted against V_t and is shown in Fig 4C. In NZs and IZs, V_0.5 was -30.1±0.5 mV (n=54) and -29.1±0.7 mV (n=36), respectively, with slope factors of k=6.99±0.2 and 7.54±0.2 (P>.05). Estimated E_rev was -0.4±0.4 mV in NZs and -1.0±0.5 mV in IZs (P<.05).

View larger version (26K): [in this window] [in a new window]   Figure 4. Steady state inactivation and activation of INa in NZs and IZs. The original tracings of INa obtained during the "steady state" inactivation (I/Imax) protocol in a typical NZ (V0.5=-80.2 mV, 124 pF; A) and a typical IZ (V0.5=-83.6 mV, 109 pF; B) after subtraction of membrane capacity and linear leakage. Arrows indicate INa after various conditioning potentials. Note that the amplitude of INa in NZ was reduced to half after a conditioning potential to -80 mV, whereas in the IZ, half-amplitude occurred at -85 mV. Panel C, left side, shows average I/Imax curves constructed using data recorded with the same protocol in both cell types. The current amplitude elicited by the test pulse at each prepulse voltage was normalized to the maximal current obtained after prepulse voltages to -120 mV. The I/Imax curve was created by plotting the normalized current against Vc. V0.5 was -80.2±0.48 mV in NZs () and -83.9±0.59 mV in IZs () (P<.01). The averaged time after membrane rupture was 25.2±0.6 minutes in NZs vs 25.9±0.7 minutes in IZs (P<.05). Note that at -100 mV, the availability of INa was 97.5±0.35% in NZs and 92.9%±1.2% in IZs. Panel C, right side, shows the average activation curves constructed using data derived from I-V relationships for all cells. The averaged V0.5 value was -30.1±0.52 mV in NZs and -29.1±0.66 mV in IZs (P>.05). Panel D shows an expanded plot of data in panel C, illustrating a small "window" current or overlap area of inactivation and activation curves of NZs vs IZs.

In order to examine the effects of altering the duration of the conditioning prepulse on I_Na, we examined the effects of prepulse duration on the I/I_max curve in a subset of NZs (n=15) and IZs (n=14). In these cells, the maximally available I_Na with prepulse of 1 s was significantly reduced in IZs compared with NZs. An increase in duration of prepulse shifted the I/I_max curve in a hyperpolarizing direction and decreased the slope factor in a similar manner in both NZs and IZs (Table 2). Thus, by prolonging the prepulse duration, we did not observe an exaggeration of differences between the two groups.

View this table: [in this window] [in a new window]   Table 2. Steady State Inactivation With Varied Prepulse Duration

Inactivation of Na⁺ Channel From a Membrane Potential Negative to Current Activation
It is known that the cardiac Na⁺ channel may inactivate directly from a closed state; ie, inactivation of the channel can occur without opening.^{28 32} To examine this process in cells within each group, a conditioning prepulse to -60 mV (subthreshold voltage) of increasing duration was followed by a 2-ms interval to V_H=-100 mV and then a subsequent test pulse to -25 mV. Fig 5A shows that in NZs, I_Na amplitude is reduced as the duration of conditioning prepulse is increased, indicating that more and more Na⁺ channels become inactivated as the time interval at -60 mV is increased. However, in the IZ (Fig 5B), I_Na is reduced to half the original amplitude after only a 50-ms prepulse, whereas in the NZ (Fig 5A), it took close to a 100-ms prepulse to reach approximately half the current amplitude. Clearly, with a prepulse of 300 ms, little or no I_Na remains in the IZ, but nearly 300 pA is still available in the NZ. For comparison, the amplitude of peak I_Na elicited by each test pulse was normalized to peak I_Na (1-ms prepulse) and plotted against the duration of the prepulse. A biexponential function was used to fit data to describe the time course of development of inactivation for each cell. Both ₁ and ₂ of these relationships were significantly smaller in IZs compared with NZs (Table 3). Data used to construct these curves were obtained at similar times after membrane rupture (NZs, 34.3±5 minutes; IZs, 38.4±6 minutes; P<.05). Thus, Na⁺ channels existing in IZs appear to inactivate from a closed state faster than those of NZs.

View larger version (19K): [in this window] [in a new window]   Figure 5. Rate of development of inactivation of INa from depolarized conditioning potential (-60 mV). Capacity and leak-subtracted INa tracings from typical NZ (A) and IZ (B) obtained during the protocol are shown. In both panels, test INa (VH=-100 to -25 mV) obtained after prepulses to -60 mV of variable duration are superimposed so as to emphasize differences in inactivation between cells from two groups. Note that in IZ, INa amplitude is reduced to half only after a conditioning pulse of 50 ms (arrow), whereas in the NZ, a conditioning pulse of 85 ms (arrow) is needed to inactivate half of INa. In panel C, averaged results are plotted for NZs () and IZs (). For the graph, INa amplitude of the test pulse is normalized to maximal INa (Imax, obtained with 1-ms conditioning pulse). The time course of INa inactivation was best described by a biexponential function (for data see Table 3). Inset shows initial part of relationships on an expanded time scale. Note the difference in time to reach 50% of Imax in IZs versus NZs. Cell membrane capacity was as follows: NZ=124 pF and IZ=147 pF.

View this table: [in this window] [in a new window]   Table 3. Time Constant of INaInactivation After Prepulse to -60 mV

Recovery From Inactivation
The time course of recovery of I_Na from steady state inactivation was studied using a conventional double-pulse protocol. Inactivation was induced by a 1-s prepulse (-100 to -25 mV), followed by a test pulse to -25 mV after IPI that varied from 2 to 3000 ms. The pulse pair was repeated every 5 s. Three levels of V_H (-90, -100, and -110 mV) were used in this series of experiments. Recovery was accelerated when V_H was hyperpolarized in both cell groups. The time course of recovery from inactivation was significantly slower in IZs than in NZs at V_H=-100 and -110 mV (Table 4). At V_H=-90 mV, I_Na recovered after an initial lag in 18 (82%) of 22 IZs. This lag was observed in only 2 (9%) of 22 NZs. Fig 6A shows normalized I_Na plotted against IPI for V_H=-90 and -110 mV in an NZ and IZ, respectively. Panels B and C of Fig 6 illustrate the initial process of the recovery of I_Na in these two cells. Note that in the IZ, an initial lag of I_Na recovery was observed when V_H was -90 mV (see also inset). The time constants of recovery of I_Na were V_H dependent (Table 4).

View this table: [in this window] [in a new window]   Table 4. Time Constants of Recovery of INa From Inactivation

View larger version (28K): [in this window] [in a new window]   Figure 6. Time course of recovery of INa in typical NZ and IZ. Plots of data were obtained using the double-pulse protocol (see text). The amplitude of INa at each IPI is normalized to INa at IPI=3000 ms. Panel A illustrates recovery in a typical NZ (circle) and IZ (square) at the two different VH values (VH=-110 mV [unfilled symbol] and VH=-90 mV [filled symbol]). Inset graph shows average data obtained in two cell groups (NZs, ; IZs, ) for short IPIs for VH=-90 mV. Panels B and C illustrate that recovery of INa depended on VH in a typical NZ (B) and IZ (C). Note the lag in recovery of INa seen in the IZ when VH=-90 mV. Data were best described biexponentially in both cell types. Data used to construct these relationships were obtained at similar times after membrane rupture (NZs: -90 mV at 31±6 minutes, -100 mV at 31±6 minutes, and -110 mV at 35±6 minutes; IZs: -90 mV at 32±4 minutes, -100 mV at 31±5 minutes, and -110 mV at 38±5 minutes).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We found that there is a significant reduction (61%) of the peak I_Na density in myocytes surviving in the epicardial border zone 5 to 6 days after total coronary occlusion. This result is consistent with the previous finding from our laboratory¹¹ in which _max of the action potential was found to be significantly reduced in IZs. Patterson at al¹² also reported similar results using action potential recordings of myocytes of EBZ 4 days after myocardial infarction.

Additionally, although the voltage dependence of activation of I_Na and the time course of decay of peak I_Na were no different between groups, we found that the average I/I_max curve was shifted significantly in the hyperpolarizing direction and that the inactivation time course of I_Na from a depolarized prepulse potential was significantly accelerated in IZs versus NZs. These results are consistent with the myocyte results using _max^{11 12} as well as with I_Na results from human atrial cells under conditions that mimic ischemia.³³ A negative shift of the availability curve for I_Na in IZs may be one of the contributors to the reduced _max observed in normal resting potential myocytes that survive in the infarcted heart^{11 12} and could account for a reduced Na⁺ "window" current that would be important during the plateau phase of the action potential. A reduction in Na⁺ window current is consistent with the "triangularization" of the action potentials of myocytes surviving in EBZ 5 days after coronary artery occlusion.^{9 10 11}

The fact that with a small membrane depolarization, I_Na inactivates faster in IZs than in NZs suggests that the Na⁺ channel inactivation without channel opening may be enhanced in IZs. In the present study and in others,^{27 28} two exponentials are necessary to describe this process in both cell types. This may mean that there exist two different paths leading to the inactivated states of the Na⁺ channels in control cells and that IZs prefer to enter these states. Enhanced inactivation of I_Na in IZs at a depolarized potential could have a significant impact on the modulation of I_Na availability (membrane excitability) of IZs at rest in the beating heart and may contribute to the abnormal conduction properties of the myocytes in the in situ EBZ.^{9 17 34}

Another major finding was the significant difference in the time course of recovery of I_Na from inactivation between NZs and IZs. The recovery process was voltage dependent in both cell types but was significantly slower in IZs. At the most depolarized repriming voltage, I_Na recovery showed a lag before onset in most IZs but not in NZs. These results are consistent with our findings in which the recovery of _max was slower and had a lag before onset in IZs compared with NZs.¹¹ Interestingly, the contribution of either the fast or slow component to the total recovery time course of I_Na remained unchanged under our recording conditions.

In the present study and in others,^{26 27 29} I_Na recovery from inactivation has been determined to have at least two components. In most studies on I_Na in normal myocytes, a lag in the onset of recovery of I_Na from inactivation has not been described. However, in a recent study,²⁹ adult rat I_Na exhibited a lag before onset of recovery only when the repriming voltage was set to 70% availability. However, the magnitude of the lag observed in these latter experiments was only 0.5 to 3 ms, less than what we observed in two NZs under our study conditions. In normal cat atrial cells, the lag before the onset of I_Na recovery was reported to be 6 ms, again similar to our NZ values.²⁷ In IZs, at the most depolarized repriming potential (70% availability), significant lags were observed in most cells. Kuo and Bean³⁵ have suggested that in rat CA1 neurons the lag in onset of recovery of I_Na from inactivation is related to a lag in channel deactivation. If this scheme is correct for the cardiac I_Na, it suggests then that in IZs Na⁺ channels may deactivate slowly, before the "unbinding" of the inactivation occurs. It is also possible that there are two populations of cells, each having different recovery kinetics, and that in IZs there is a larger percentage of the more slowly recovering cells. The slower recovery of I_Na or recovery of I_Na after a lag may contribute to slowed conduction or prolongation of refractoriness (postrepolarization) of fibers of EBZ of the infarcted heart.^{9 13 36} However, other changes, such as redistribution and altered function of gap junctions in the EBZ,³⁷ may also contribute to the arrhythmic properties of the EBZ.

Experimental Considerations
Experimental conditions of these experiments were highly unphysiological yet chosen for the following reasons. First, voltage-clamp control of the cell membrane was considered. Under physiological conditions, I_Na is so large and the kinetics are so rapid that successful voltage-clamp control is challenging, even in single-cell preparations.^{31 38} However, some investigators have been successful using a macropatch technique.^{39 40} For these reasons, the usual way to study I_Na in whole-cell recording configuration is to reduce the experimental temperature and lower the Na⁺ gradient.^{26 30 31 41 42} Under our conditions, I_Na in 91% of NZs was reduced to <3 nA, with the largest I_Na being 4.16 nA. These values are similar to others under similar conditions.^{6 26 40} It could be that gating changes have occurred under this reduced Na⁺ concentration, resulting in our findings. However, we studied NZs and IZs under identical conditions; therefore, to understand our results, we would have to assume that this effect was enhanced in IZs. We think that this is unlikely. Furthermore, it appears that just the reduction in density of I_Na cannot account for the observed kinetic changes, since kinetic changes in I_Na were not observed in NZs with densities similar to those of IZs (data not shown). Additionally, to enhance voltage-clamp control, large patch pipettes were used such that the time constant of decay of the capacitive transient averaged 160 µs and the time to peak current values were similar. Thus, averaged steady state voltage error resulting from residual series resistance was 2 mV, similar to that found in other studies.^{26 43 44} The absence of crossover of the current traces as the current magnitude was changed and the I-V relationship spanning 30 mV in the negative limb in our data further suggest adequate voltage control.^{25 26} Thus, the clamp voltage was properly controlled in our dialyzed cell experiments, and it is unlikely that our findings of reduced I_Na result from clamp conditions.

A time-dependent negative shift of the steady state I/I_max curve may contribute to our finding of reduced I_Na in IZs; however, we think it unlikely for the following reasons: A time dependent change in current amplitude and shift of I/I_max curve during whole-cell recording has been described by many. Subsequently, Hanck and Sheets²⁴ reported a -0.41-mV/min shift of I/I_max curves of canine cardiac Purkinje cells after membrane rupture. These reported time-dependent changes are faster than our measured rates (0.14 and 0.11 mV/min in NZs and IZs, respectively). Nevertheless, we found time-dependent changes to be similar in cells of the two groups and thus always compared data from cells of the two groups at similar times after membrane rupture.

Finally, we think it unlikely that the current reduction and alteration of Na⁺ channel kinetics are due to the surgical procedure, cell isolation process, or criteria used for cell selection, as we have discussed previously.^{11 20} A contamination of outward currents present only in IZs cannot account for the I_Na reduction, since the composition of intracellular and extracellular solutions was chosen to minimize contaminating currents. An increase of the cell capacitance or membrane surface area without change in the absolute channel number and a negative shift of I/I_max curve can be responsible for only a portion of the current reduction measured, since the total amplitude of I_Na was also reduced in IZs versus NZs, and the maximally available I_Na (determined at V_c=-120 mV) remained significantly different in IZs versus NZs. Therefore, there must be other factors responsible for the diminished I_Na density and altered Na⁺ channel kinetics in IZs.

Possible Mechanism of Reduction of I_Na Density and Alteration of Na⁺ Channel Kinetics
We know that the intracellular milieu is constantly changing after myocardial ischemia or infarction (see review in Reference 45⁴⁵ ). These abnormalities could affect ionic current function; however, the intracellular milieu of cells from both groups was equilibrated with a similar pipette solution during our whole-cell recordings. Thus, the abnormalities of I_Na function that we observed in IZs are most likely due to a chronic change in ion channel function secondary to the myocardial infarction and not due to changes in the ion content of the intracellular milieu in IZs.

The cytoskeleton, which has been shown to play an important role in the maintenance of cell structure and function,⁴⁶ is also involved in the regulation of voltage-dependent Na⁺ channel function.⁴⁷ These effects have been attributable to the channel entering a mode of reduced peak open probability in which long bursts of openings occurred.⁴⁷ After acute ischemia or myocardial infarction, disintegration or breakdown of the cytoskeleton has been documented in energy-depleted cells⁴⁸ and thus may account for some but not all I_Na abnormalities observed in IZs.

Na⁺ channel number and mRNA levels in the heart can be modulated by intracellular Ca²⁺.^{49 50} Recently, using whole-cell I_Na and single-channel recordings from cultured neonatal rat cells, Chiamvimonvat et al⁵¹ investigated the regulation of the function of Na⁺ channel biosynthesis by cytosolic Ca²⁺ and showed that I_Na density was significantly decreased in cells with high levels of intracellular Ca²⁺ and increased in cells exposed to BAPTA AM, or cells with low levels of intracellular Ca²⁺. However, in these studies, the voltage dependence of activation and inactivation and single-channel conductance of I_Na were not changed, suggesting that the differences in I_Na observed were secondary to a change in Na⁺ channel number rather than to a change in single-channel conductance or gating. Intracellular Ca²⁺ may well increase after myocardial infarction; thus, this persistent elevation of intracellular Ca²⁺ may inhibit Na⁺ channel synthesis. Our data that IZs have reduced I_Na density and amplitude suggest the possibility that the Na⁺ channel number is reduced. However, increased intracellular Ca²⁺ alone cannot account for all changes, since the reduced I_Na in IZs also recovered more slowly and exhibited altered inactivation properties.

Physiological and Pharmacological Implications
Reduced I_Na and altered Na⁺ channel kinetics in IZs may have important physiological and pharmacological implications, since I_Na not only generates the action potential upstroke but also regulates the action potential duration. Furthermore, Na⁺ channels are often the target of antiarrhythmic drugs. From this point of view, altered Na⁺ channel function in IZs may show a change in sensitivity to antiarrhythmic drugs that block Na⁺ channels, such as lidocaine. Some in vitro studies using the multicellular EBZ preparation have suggested that lidocaine preferentially depressed conduction and prolonged refractoriness in EBZ versus control fibers.^{15 52} On the other hand, reduced Na⁺ channel function in IZs may be responsive to Na⁺ channel activator agents, which could be useful in this setting. For example, BDF 9148, a known cardiac inotropic agent,⁵³ appears to be less arrhythmogenic than norepinephrine or ouabain.⁵⁴ How to restore ion channel function in myocytes that survive in the infarcted heart remains to be investigated.

	Selected Abbreviations and Acronyms

 

1, 2 = fast and slow time constants EBZ = epicardial border zone Erev = reversal potential gmax = max gNa gNa = normalized conductance of INa I-V = current-voltage I/Imax curve = availability curve INa = Na+ current IPI = interpulse interval IZ = myocyte from EBZ k = slope factor LAD = left anterior descending coronary artery NZ = myocyte from noninfarcted epicardium V0.5 = half-maximal activation voltage Vc = conditioning voltage VH = holding potential Vt = test voltage

	Acknowledgments

 
This study was supported by grant HL-30557 from the National Heart, Lung, and Blood Institute, Bethesda, Md.

Received August 6, 1996; accepted May 1, 1997.

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