Extracellular proton depression of peak and late Na⁺ current in the canine left ventricle

Abstract

Cardiac ischemia reduces excitability in ventricular tissue. Acidosis (one component of ischemia) affects a number of ion currents. We examined the effects of extracellular acidosis (pH 6.6) on peak and late Na⁺ current (I_Na) in canine ventricular cells. Epicardial and endocardial myocytes were isolated, and patch-clamp techniques were used to record I_Na. Action potential recordings from left ventricular wedges exposed to acidic Tyrode solution showed a widening of the QRS complex, indicating slowing of transmural conduction. In myocytes, exposure to acidic conditions resulted in a 17.3 ± 0.9% reduction in upstroke velocity. Analysis of fast I_Na showed that current density was similar in epicardial and endocardial cells at normal pH (68.1 ± 7.0 vs. 63.2 ± 7.1 pA/pF, respectively). Extracellular acidosis reduced the fast I_Na magnitude by 22.7% in epicardial cells and 23.1% in endocardial cells. In addition, a significant slowing of the decay (time constant) of fast I_Na was observed at pH 6.6. Acidosis did not affect steady-state inactivation of I_Na or recovery from inactivation. Analysis of late I_Na during a 500-ms pulse showed that the acidosis significantly reduced late I_Na at 250 and 500 ms into the pulse. Using action potential clamp techniques, application of an epicardial waveform resulted in a larger late I_Na compared with when an endocardial waveform was applied to the same cell. Acidosis caused a greater decrease in late I_Na when an epicardial waveform was applied. These results suggest acidosis reduces both peak and late I_Na in both cell types and contributes to the depression in cardiac excitability observed under ischemic conditions.

acute myocardial ischemia is associated with a number of changes, such as a progressive shortening of the cardiac action potential (AP), a slowing of electrical conduction, and eventual loss of excitability. This loss of excitability tends to occur in ventricular epicardial (Epi) versus endocardial (Endo) tissues (12, 15, 21, 24). Several mechanisms for this Epi versus Endo tissue response have been described. The spike and dome morphology of the Epi AP facilitates loss of the AP dome during ischemia and may contribute to this differential response (27). Other studies (13, 25) have also shown ATP-sensitive K⁺ current (I_KATP) activation was greater in Epi cells when exposed to ischemic conditions and thus may contribute to the depression of excitability. More recently, it has been demonstrated that Na⁺ channels of Epi cells exhibit a more negative steady-state inactivation compared with Endo cells (1, 9), suggesting that, during depolarization, Epi cells lose Na⁺ channel availability (and excitability) before Endo cells.

During acute cardiac ischemia, a number of pathophysiological changes have been observed, such as depletion of high-energy phosphates (32, 37) and acidosis due to a reduction in intracellular and external pH (23), both which can directly affect the function of a number of ion channels (for a review, see Ref. 4). A number of studies have examined the extent by which external pH is reduced during cardiac ischemia. In rat hearts subjected to 20 min of global ischemia, a dramatic reduction in extracellular pH was observed (from pH 7.3 to 5.5), as assessed by nuclear MRI (14). A similar reduction in extracellular pH was observed in rabbit hearts subjected to global ischemia. Using pH-sensitive electrodes, Weiss et al. (36) found that external pH was 7.3 under control conditions and decreased 0.75 log units after 10 min of ischemia.

Ion channel function is known to be altered by acidosis (one component of ischemia). Changes in the magnitude and gating of L-type Ca²⁺ current (5), rapid delayed rectifier K⁺ current (10), and transient outward K⁺ current (I_to) (16) have all been reported during acidosis. Interestingly, when Epi and Endo cells from the rat ventricle were exposed to acidosis, a differential effect on APs and K⁺ currents was observed (22). The results of that study suggest there are cell-specific responses to a reduction in external pH, presumably due to the different complement of K⁺ currents between Epi and Endo cells. Since intrinsic differences in Na⁺ channel function between Epi and Endo cells exist (9), it suggests that acidosis would produce a differential response in Na⁺ channel function as well.

In this study, we examined the effect of acidosis on both peak and late Na⁺ current (I_Na) recorded from Epi and Endo cells isolated from the canine left ventricle. Since Weiss et al. (36) reported a modest reduction (0.75 log units) in extracellular pH after 10 min of ischemia, we chose an external pH of 6.6 for our experiments. The results of our study show that acidosis reduces both peak and late I_Na in both cell types without affecting steady-state inactivation or recovery from inactivation. Using AP voltage-clamp recordings of late I_Na, we found a larger late I_Na when an Epi waveform was applied to cells. Our observations suggest that extracellular protons (one component of ischemia) directly interact with Na⁺ channels, resulting in a reduction of both peak and late I_Na and contributing to the loss of excitability during ischemia. Preliminary results have been presented in abstract form (31).

METHODS

Ventricular wedge preparations.

Adult mongrel dogs of either sex were used for all experiments, and all animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1985) and were approved by the Animal Care and Use Committee of the Masonic Medical Research Laboratory. Animals were anticoagulated with heparin and anesthetized with pentobarbital (30–35 mg/kg iv). The chest was open via a left thoracotomy, and the heart was excised, placed in cardioplegic solution (4°C Tyrode solution with 12 mM extracellular K⁺ concentration), and transported to a dissection tray. Transmural wedges with dimensions of up to 3 × 2 × 1.5 cm (left ventricular wedges) were dissected from the anteroapical aspects of the canine left ventricle. During the cannulation procedure, the preparations were initially arterially perfused with cardioplegic solution through a distal diagonal branch of the left anterior descending coronary artery. Subsequently, the wedges 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% O₂-5% CO₂ (37 ± 0.5°C). The perfusate was delivered at a constant pressure (45–50 mmHg). A transmural ECG was recorded using two Ag/AgCl half-cells placed at ∼1 cm. from the Epi (+) and Endo (−) surfaces of the preparation and along the same axis as the transmembrane recordings. APs were simultaneously recorded from the Epi surface and from sub-Endo regions or the Endo surface using floating microelectrodes. Pacing was applied to the endocardial surface [basic cycle lengths (BCLs) = 300–800 ms]. All amplified signals were digitized and analyzed using Spike 2 for Windows (Cambridge Electronic Design, Cambridge, UK).

Isolated myocyte preparation.

Myocytes from Epi and Endo regions were prepared from canine hearts using previously described techniques (6, 8). A wedge consisting of the left ventricular free wall supplied by a descending branch of the circumflex artery (left marginal artery) was excised, cannulated, and perfused with nominally Ca²⁺-free Tyrode solution containing 0.1% BSA for a period of ∼5 min. The wedge preparations were then subjected to enzyme digestion with the 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 the Epi (<2 mm from the Epi surface) and Endo (<2 mm from the Endo surface) were shaved from the wedge using a dermatome. The tissue slices were then placed in separate beakers, minced 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 myocytes was stored at room temperature.

AP recordings from single myocytes.

APs from ventricular cells were recorded using whole cell patch pipettes coupled to a MultiClamp 700A amplifier (Axon Instruments, Foster City, CA) as previously described (11). Briefly, cells were superfused with HEPES buffer of the following composition (mM): 126 NaCl, 5.4 KCl, 1.0 MgCl₂, 2.0 CaCl₂, 10 HEPES, and 11 glucose. pH adjusted to 7.4 with NaOH. For acidosis experiments, HEPES buffer of pH 6.6 was obtained by the addition of HCl. The patch pipette solution had the following composition (in mM): 90 K-aspartate, 30 KCl, 5.5 glucose, 1.0 MgCl₂, 5 EGTA, 5 MgATP, 5 HEPES, and 10 NaCl (pH 7.2 with KOH). The resistance of the electrodes was 2–4 MΩ when filled with the pipette solution. APs were elicited using a 3-ms current pulse at 120% threshold amplitude, and cells were paced at cycle lengths of 0.5 and 1 Hz. Acidic buffer was rapidly applied using a quartz micromanifold (ALA Scientific, Farmingdale, NY) placed in close proximity to the cell. APs were acquired at 50 kHz and filtered at 5 kHz.

Voltage-clamp recordings of peak I_Na.

Early I_Na was measured as previously described (9) with minor modifications. Experiments were performed using a MultiClamp 700A (Axon Instruments). Command voltages were delivered, and data were acquired via a DigiData 1322 computer interface using the pCLAMP 9 program suite (Axon Instruments) with data stored on 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 0.9–2.0 MΩ when filled with the internal solution (see below). The membrane was ruptured by applying negative pressure and series resistance 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, 1.5 MgCl₂, 1 CoCl₂, 10 glucose, 10 HEPES, 5 NaOH, and 0.1 BaCl₂ (pH adjusted to 7.4 with NaOH/HCl). Low-Na⁺ external buffer of pH 6.6 was obtained by the addition of HCl. The pipette solution contained (in mM) 15 NaCl, 120 CsF, 1 MgCl₂, 5 KCl, 10 HEPES, 4 Na₂ATP, and 10 EGTA (pH adjusted to 7.2 with CsOH). Peak I_Na was dramatically reduced in the low extracellular Na⁺ solution to ensure adequate voltage control, as gauged by the slope of a Boltzmann fit to the steady-state activation curve (20). When measuring Na⁺ channel kinetics and density, the holding potential was −120 mV to recruit all available Na⁺ channels. In addition, recordings of I_Na were made at least 5 min after rupture to minimize the effects of the time-dependent negative shift of steady-state inactivation that occurs in conventional voltage-clamp experiments. As described above, acidic buffer was rapidly applied to cells using a quartz micromanifold. Whole cell currents were analyzed using the Clampfit analysis program from pCLAMP 9 (Axon Instruments).

Voltage-clamp recordings of late I_Na.

Late I_Na density was a measured in full external Na⁺ at 37°C as previously described (41, 42). The external solution contained (in mM) 140 NaCl, 2.0 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). The pipette solution contained (in mM) 10 NaCl, 130 aspartate, 1 MgCl₂, 10 CsCl, 10 HEPES, 5 MgATP, and 10 EGTA (pH adjusted to 7.2 with CsOH).

Late I_Na density was recorded in cells that were held at −80 mV. To remove steady-state inactivation and recruit all Na⁺ channels, a pulse to −120 mV was applied before a 500-ms pulse to −40 mV. External pH was rapidly changed to 6.6, and the protocol was repeated, followed by the rapid application of 10 μM TTX. Late I_Na, characterized as the TTX-sensitive difference current, was measured at various time intervals throughout the 500-ms step to −40 mV.

Statistics.

Results from pooled data are presented as means ± SE. Statistical analysis was performed using ANOVA followed by a Student-Newman-Keuls test or a Student t-test, as appropriate, using SigmaStat software. P values of <0.05 were considered statistically significant.

RESULTS

As an initial basis of comparison, APs and the corresponding ECG were simultaneously recorded in Epi and Endo layers from a canine left ventricular wedge preparation. Figure 1A shows AP recordings (top and middle traces) and the corresponding ECG (bottom trace) from a wedge preparation paced at a BCL of 2,000 ms. After the exposure to acidic Tyrode solution (30 min), there was a slight prolongation of the QRS complex associated with transmural conduction slowing (delayed excitation of the Epi layer). A similar slowing was observed in three other left ventricular wedge preparations. The slowing of conduction across the ventricular wall after the exposure to acidic Tyrode solution suggests 1) a reduction in Na⁺ channel current and/or 2) an increase in gap junctional resistance between neighboring cells (40). We next explored whether acidosis directly affected I_Na by measuring upstroke velocity (dV/dt) in single ventricular myocytes. APs were elicited by injection of a 3-ms current pulse, and dV/dt (an index of I_Na) was measured at pH 7.4 and 6.6. The upstroke of the AP and corresponding dV/dt from an Epi cell are shown in Fig. 1B. In cells paced at a BCL of 2,000 ms, exposure to acidic buffer reduced dV/dt to 83.7 ± 0.93% of control (n = 6). Similarly, in cells paced at a BCL of 1,000 ms, exposure to acidic buffer reduced dV/dt to 84.3 ± 2.6% of control (n = 6), suggesting that acidosis results in a reduction in the magnitude of I_Na.

Fig. 1.

A (from top to bottom): subendocardial (sub-Endo) and epicardial (Epi) action potentials (APs) and the transmural pseudo-ECG (ECG). a, Control; b, recordings obtained after 25–30 min of perfusion with low-pH Tyrode soluiton (pH 6.6); a & b, superimposed traces of the control and pH 6.6 recordings. The inset numbers on the APs indicate (in ms) the AP duration at 90% repolarization (APD₉₀). The inset numbers on each ECG tracing indicate (from left to right) the R wave width and QT interval intervals (in ms). Basic cycle length (BCL) = 2,000 ms. B: representative recordings of the AP upstroke and corresponding upstroke velocity (dV/dt) recorded from a myocyte at pH 7.4 and 6.6.

We (9) have previously shown that there are intrinsic differences in I_Na between Epi and Endo cells and speculated if acidosis can exacerbate these differences. In the next series of experiments, peak I_Na was measured in low extracellular Na⁺ buffer to ensure adequate voltage control. Recordings were made from myocytes exposed to external pH 7.4 and after the rapid application of acidic buffer (pH 6.6). We first compared the effects of acidosis on the current-voltage relationship in Epi and Endo cells (Fig. 2). Representative I_Na traces recorded from an Endo cell exposed to external pH 7.4 and then 6.6 are shown in Fig. 2, A and B, respectively. Exposure to acidic conditions resulted in a decrease in the magnitude of the current. Analysis of the current-voltage relation showed that extracellular acidosis decreased peak I_Na from −68.1 ± 7.01 to −52.5 ± 5.88 pA/pF in Epi cells (Fig. 2C) and from −63.1 ± 7.07 to −48.7 ± 5.93 pA/pF in Endo cells (Fig. 2D). To better determine the reduction in I_Na in response to acidosis, we expressed the pH 6.6 results as fractional current at each voltage for both Epi (Fig. 2E) and Endo (Fig. 2F) cells. The results show that acidosis produced a clear reduction in the size of the current at almost all voltages examined. We next determined if this reduction in peak I_Na was due to changes in steady-state activation. Chord conductance was determined using the ratio of current to the electromotive potential for the cells shown in Fig. 2, C and D, and a Boltzmann curve was fit to the normalized data. Analysis of pH effects on steady-state activation in Epi cells (Fig. 2G) showed half-activation voltages (V_1/2) of −45.1 ± 0.19 mV at pH 7.4 and −43.4 ± 0.20 mV at pH 6.6 (n = 10). For Endo cells (Fig. 2H), V_1/2 was −40.7 ± 0.21 mV at pH 7.4 and −38.7 ± 0.29 mV at pH 6.6 (n = 9).

Fig. 2.

A and B: representative whole cell current recordings from a left ventricular Endo myocyte exposed to external pH 7.4 (A) and 6.6 (B). Current recordings were obtained at test potentials between −80 and 10 mV in 5-mV increments from a holding potential of −120 mV. For each cell, the current-voltage (I-V) relation was recorded at the two different pH values. C: I-V relation for Epi cells (n = 10) showing a significant reduction in Na⁺ current (I_Na) magnitude. D: I-V relation for Endo cells (n = 9) also showing a significant reduction in I_Na magnitude. E and F: acidosis data expressed as the fractional reduction in current at each voltage for Epi (E) and Endo (F) cells. G and H: steady-state activation relation for Epi (G) and Endo (H) cells. Chord conductance was determined using the ratio of current to the electromotive potential for the cells shown in C and D. Data were normalized and plotted against their test potential. *P < 0.05 vs. control (pH 7.4).

The reduction in peak I_Na due to acidosis may be due to a shift in Na⁺ channel availability. Therefore, we next evaluated steady-state inactivation at pH 7.4 and 6.6. Peak current after a 500-ms prepulse was normalized to the maximum current and plotted as a function of the prepulse voltage, and a Boltzman function was fitted to the data. Figure 3 shows representative traces recorded from an Endo cell exposed to external pH 7.4 (A) and 6.6 (B). Acidosis did not change the mid-inactivation potential in either Epi (Fig. 3C) or Endo (Fig. 3D) cells. For Epi cells, V_1/2 in pH 7.4 and 6.6 was −81.2 ± 0.2 and −81.4 ± 0.3 mV, respectively; for Endo cells, V_1/2 in pH 7.4 and 6.6 was −71.8 ± 0.6 and −73.1 ± 0.7 mV, respectively. These results show that the decrease in peak I_Na in either cell type was not due to a shift in steady-state inactivation.

Fig. 3.

A and B: representative steady-state inactivation recordings from an Epi cell exposed to external pH 7.4 (A) and external pH 6.6 (B). The voltage-clamp protocol is shown at the top. Peak currents were normalized to their respective maximum values and plotted against the conditioning potential. C and D: the steady state-inactivation relation for Epi (C) and Endo (D) cells was unaffected by the reduction in external pH.

In the next series of experiments, we determined if I_Na recovery from inactivation was altered by acidosis. Recovery was determined using a standard double pulse protocol separated by selected time intervals (voltage-clamp protocol shown at the top of Fig. 4). Figure 4 shows representative traces recorded from an Endo cell showing I_Na recovery at pH 7.4 (A) and 6.6 (B). Reactivation of I_Na at pH 7.4 for both cell types exhibited both a fast and slow phase of recovery [time constant 1 (τ₁) and 2 (τ₂)] as follows: 1) τ₁ = 8.8 ± 0.8 ms and τ₂ = 32.6 ± 1.9 ms for Endo cells and 2) τ₁ = 7.5 ± 0.6 ms and τ₂ = 31.2 ± 2.2 ms for Epi cells. Recovery was not significantly affected by acidosis in either cell types (Fig. 4, C and D).

Fig. 4.

A and B: traces recorded from an Endo cell exposed to external pH 7.4 (A) and 6.6 (B). Recovery was measured using two identical voltage-clamp steps to −20 mV from a holding potential of −100 mV separated by selected time intervals. C and D: the recovery time course of I_Na recorded from Epi (C) and Endo (D) cells was unaffected by acidosis.

Peak I_Na density was significantly reduced by acidosis in both cell types. In addition, the decay of I_Na appeared to be slowed. Figure 5A shows the time course of changes in I_Na from a left ventricular Epi myocyte exposed to external pH 7.4 (black trace) and 6.6 (red traces). The cell was activated 100 times at a rate of once per second, and every 10th pulse is shown. Acidosis produced a rapid decrease in the size of I_Na as well as a slowing of the time to peak and decay of I_Na. This effect reached steady state after about the 40th pulse, since pulses 40–100 were virtually superimposable. To better quantify, we next analyzed the decay of the current by analyzing the inactivation kinetics of I_Na at pH 7.4 and 6.6 in both cell types. The decay of I_Na (current traces are shown in Fig. 2) elicited by pulses positive to −40 mV was fit with a monoexponential function. Under control conditions, the time constant of decay was significantly slower in Endo cells compared with Epi cells (solid circles in Fig. 5, B and C). After exposure to pH 6.6, the time constant of decay was significantly slowed in both cell types.

Fig. 5.

A: representative whole cell current recordings showing the time course of changes in I_Na from a left ventricular Epi myocyte exposed to external pH 7.4 (black trace) and 6.6 (red traces). The cell was activated 100 times at a rate of once per second, and every 10th pulse is shown. Acidosis produced a rapid decrease in the size of I_Na as well as a slowing of the decay. This effect reached steady state after about the 40th pulse. Inactivation time constants (τ) for I_Na decay as a function of voltage are shown. Inactivation τ values were measured by fitting a single-exponential function to the current decay. B: inactivation τ values of I_Na for Epi cells as a function of voltage. *P < 0.05 vs. control (pH 7.4). C: inactivation τ values of I_Na for Endo cells as a function of voltage. *P < 0.05 vs. control (pH 7.4).

An increase in persistent or late I_Na has been observed in response to ischemia and hypoxia (19, 34). Since the decay of peak I_Na (during the 25-ms test pulse) was slowed in response to acidosis, we wondered if the slowing of decay resulted in a larger persistent or late current during acidosis. We next measure late I_Na during a 500-ms test pulse at pH 7.4 and 6.6. Late I_Na recordings were performed in full external Na⁺ at 37°C. In Fig. 6A, currents measured at −40 mV are shown under control conditions (pH 7.4) and after the application of 10 μM TTX. Subtraction of the TTX trace from the control trace results in the TTX-sensitive difference current shown in Fig. 6B. Similar subtractions were performed in Epi and Endo cells at pH 7.4 and 6.6, and the mean TTX-sensitive currents during intervals of 50, 250, and 500 ms after the start of the depolarizing pulse are shown (Fig. 6, C and D). Analysis of late I_Na during a 500-ms pulse showed that the acidosis significantly reduced the magnitude of late I_Na at 250 and 500 ms into the pulse. At 50 ms into the pulse, there also appeared to be a trend toward a reduction at this time point (Fig. 6, C and D).

Fig. 6.

A: representative late I_Na recorded during a train of five pulses in control solution (pH 7.4) and after the application of TTX (10 μM). B: subtraction of the traces shown in A yielded TTX-sensitive late I_Na. C: bar graph showing late I_Na density in Epi cells during the 500-ms pulse to −40 mV. D: bar graph showing late I_Na density in Endo cells during the 500-ms pulse to −40 mV. *P < 0.05 vs. control (pH 7.4).

The results thus far show that acidosis reduces peak I_Na (Fig. 2), slows the decay of I_Na, and produces a significant reduction in late I_Na (Fig. 6). To assess I_Na during the course of an AP, we evaluated late I_Na total charge by integrating the area under the current trace elicited by an AP clamp. We applied prerecorded Epi and Endo ventricular AP waveforms to the same myocyte. Representative TTX-sensitive sensitive late I_Na recordings are shown in Fig. 7. Application of an Epi waveform resulted in a significantly greater charge movement (as assessed by integrating the area under the current trace) than when an Endo waveform was applied to the same cell. The late I_Na charge movement was −207 ± 15.6 pC after the application of an Epi waveform and −188 ± 17.0 pC when an Endo waveform was applied (n = 6, P < 0.05). Reducing extracellular pH resulted in a 38% reduction in charge when Epi waveform was applied and 22% reduction when an Endo waveform was applied to the same myocyte.

Fig. 7.

A and B: representative AP voltage-clamp traces recorded after the application of an Epi (A) and Endo (B) waveform. Reducing external pH from 7.4 to 6.6. reduced the total charge (as assessed by integrating the area under the current trace).

DISCUSSION

To our knowledge, this is the first study to examine the effects of acidosis on persistent or late I_Na. The results of our study demonstrate that a reduction in external pH reduces the size of peak I_Na and slows the rate of decay. The slowing did not result in a greater persistent current as late current was also reduced. Acidosis did not affect steady-state inactivation or recovery from inactivation. Using AP voltage-clamp techniques, we found that application of an Epi waveform resulted in a greater total charge compared with when an Endo waveform was applied to the same cell. Moreover, acidosis produced a greater reduction in total charge when an Epi waveform was applied. The reduction in I_Na observed in myocytes likely contributes to the slowing of conduction across the ventricular wall.

Previous studies have examined the effects of components of ischemia on ion channel function. At the cellular level, myocytes have been superfused with modified Tyrode solution designed to mimic specific conditions of ischemia. These included hyperkalemia, acidosis, hypoxia, substrate deprivation, and lactate accumulation (7, 38). Further studies have examined the various components of ischemia at the single cell level (26, 28). In this study, we examined the effects of acidosis on both peak and late I_Na. Acidosis was produced by decreasing the pH of the superfusing buffer from 7.4 to 6.6. Similar to previous investigations, we found that moderate reductions in external pH (∼1 log unit) produced about a 20% reduction in the magnitude of I_Na (39). Although an increase in persistent I_Na has been observed during ischemic conditions (19, 34), it is clear that acidosis (one component of ischemia) does not increase late I_Na.

In agreement with previous studies, we found no significant differences in the magnitude of peak I_Na (1, 9) or late I_Na (42) in Epi versus Endo cells. Interestingly, Epi cells exhibited a faster current decay and more negative steady-state inactivation compared with Endo cells. The mechanism underlying these differences is not known. Since the density of both peak (Fig. 2) and late (Fig. 6) I_Na is equivalent in Epi and Endo cells, it suggests similar amounts of the pore-forming Na_v1.5 α-subunit. The more negative half-inactivation voltage and faster current decay observed in Epi cells may be secondary to different proportions of various β-subunits (β₁–β₄) or different scaffolding proteins, all which combine to form the Na⁺ channel macromolecular complex (30).

The effect of acidosis (one component of ischemia) on the AP waveform is complex because the function of many ion channels is known to be altered. In addition, the effect of acidosis appears to be tissue specific, presumably due to the different complement of currents in the various tissue types. A reduction in the magnitude of the rapid delayed rectifier K⁺ current and L-type Ca²⁺ current was observed in atrioventricular node cells exposed to acidosis (5). In contrast, acidosis did not affect L-type Ca²⁺ current in rat ventricular myocytes (22). Interestingly, the response of I_to (16) to acidosis is complex and appears to be dependent on the resting potential of the cell. At depolarized potentials, acidosis increases the magnitude of I_to in the ventricle, whereas at more negative potentials there is little effect on this current (16, 35). These complex effects combined with the larger I_to in Epi versus Endo cells are also thought to contribute to the regional and tissue specific differences of the effects of acidosis on the AP (22).

In the present study, we observed a slowing of transmural conduction across the isolated ventricular wall leading to a slight widening of the QRS complex in the ECG upon exposure to acidosis. The slowing of conduction may be the result of many factors, such as increased gap junction resistance or a reduction in I_Na. AP recordings from single cells showed a reduction in dV/dt, suggesting that I_Na was, in part, responsible. Exposure to acidosis reduced the magnitude of peak I_Na and slowed the time to peak and decay of the current, without affecting steady-state inactivation or recovery from inactivation. These observations suggest the reduction in I_Na is due to a direct interaction of protons with the pore of the channel. Alternatively, extracellular protons may alter the charge on the side chain of certain amino acids (particularly negatively charged amino acids) that form the Na⁺ channel complex, resulting in a change in permeation. A recent preliminary report (18) found that a histidine residue at position 880 played an important role in pH modulation and function of Na_v1.5. Since the side chain of histidine residues have a pKa of 6.0, at physiologically relevant pH values, small shifts in pH will affect the charge on the side chain.

Of interest is the effect of the AP waveform on the kinetics of late I_Na and the response to acidosis. When late I_Na was measured by the application of a square-wave voltage pulse, the magnitude of current was similar in Epi and Endo cells. Furthermore, acidosis produced a similar decrease in late I_Na in both cell types. It is well established that AP waveform and duration can indirectly affect the size and kinetics of currents (2, 17, 33). Using AP clamp techniques, Magyar et al. (29) demonstrated that the application of AP waveforms of different configurations to the same TSA201 cells expressing Na_v1.5 channels dramatically altered late I_Na kinetics. In our study, application of an Epi waveform resulted in a larger late I_Na than when an Endo waveform was applied to the same cell. These observations suggest that cells with a prominent spike and dome morphology (i.e., Epi and midmyocardial cells) would have a greater contribution of late I_Na compared with cells lacking a prominent phase 1 and having a consistently high plateau (i.e., left ventricular Endo cells). These waveform-specific differences are explained by the fact that inactivation of Na⁺ channels was much faster at positive potentials. Therefore, application of an Endo waveform (with a high plateau) results in the rapid inactivation of late I_Na and less contribution of this current during the course of an Endo AP. Conversely, the presence of a spike and dome morphology, therefore, results in a less inactivation during the early phases of the AP and greater late I_Na.

Physiological implications.

During acute myocardial ischemia, electrocardiographic ST segment elevations have been well documented both clinically and experimentally. Under control conditions, AP duration is longer in Endo than Epi cells, resulting in a positive T wave in the ECG (Fig. 1A). Exposure of ventricular tissue to ischemic conditions results in a decrease in the amplitude of phase 0, a marked depression in the AP plateau (dome) recorded from Epi cells, and shortening of the AP duration (27). In contrast, ischemia produces a minor abbreviation of AP duration in Endo cells (27). This difference in repolarization gradient is manifested as an elevated ST segment in the ECG trace. Transmural differences in I_KATP, I_to, and I_Na have been shown to contribute to the differential responses of Endo and Epi cells during ischemia. In the present study, acidosis alone did not produce a sufficient reduction in I_Na to cause loss of excitability despite a modest but significant reduction in both peak (Fig. 2) and late (Fig. 6) I_Na. The reduction in peak I_Na during acidosis coupled with other components of ischemia (such as depolarization of the membrane potential) would reduce Na⁺ channel availability and excitability. Moreover, in cell types with a prominent spike and dome morphology (i.e., Epi and midmyocardial cells), it appears there is a greater contribution of late I_Na during the plateau of the AP compared with cells lacking a prominent phase 1 and having a consistently high plateau (Fig. 7). Since acidosis produced a greater reduction in late I_Na in cell types with a spike and dome morphology, this loss of depolarizing current during the plateau of the AP may also contribute to the marked depression in the AP dome from Epi cells and the ST segment elevation observed during ischemia.

Limitations of the study.

The results of our study show there are intrinsic differences in Na⁺ channel function between Epi and Endo cells, as we have previously reported (9), as well as differences in the responses of these cell types to acidosis. It should be noted that the conditions used to record peak and late I_Na were quite different. While APs and late I_Na experiments were recorded in full external Na⁺ solution at 36°C, peak I_Na had to be recorded at room temperature in greatly reduced external Na⁺ solution to ensure adequate voltage control. Therefore, the effect of protons on I_Na may be different under the two experimental conditions. It is unlikely that changing external pH would result in a reduction in intracellular pH since the pipette solution was buffered. Although bulk cytoplasmic pH likely does not change, we cannot exclude the possibility there was accumulation of protons under the sarcolemma, which may affect Na⁺ channel function (3).

GRANTS

This work was supported by American Heart Association Grant 10GRNT4210016 (to J. M. Di Diego), the American Health Assistance Foundation (to J. M. Cordeiro), and National Heart, Lung, and Blood Institute Grant HL-47678 (to C. Antzelevitch). D. Renodin was the recipient of a Summer Fellowship from the American Heart Association-Northeast Affiliate.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).