INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE TRANSIENT OUTWARD K⁺ current (I_to) is present throughout the heart of many species; it is found in atrial and ventricular muscle, the sinoatrial node, atrioventricular node, and Purkinje fibers (see Ref. 8 for review). This current is an important determinant of the early repolarization phase of the cardiac action potential, and previous studies have shown a marked heterogeneity in action potential configuration within the ventricular wall that has been ascribed to regional differences in the density of I_to. Thus the action potential has a characteristic "spike and dome" morphology in the subepicardium, where I_to is prominent, that is less marked in the subendocardium, where I_to is small or absent (3, 21).

Given its important role in determining the configuration of the cardiac action potential, and the heterogeneity in the density of I_to in different regions of the heart, interventions that modulate I_to may have important consequences on action potential dispersion and hence on the spread of the action potential through the heart. This may be important in a number of pathological conditions that have been reported to alter I_to, for example, metabolic inhibition (25) and heart failure (5). Antzelevitch et al. (3) reported that simulated ischemia caused a marked depression of the subepicardial action potential, which could be reversed by 4-aminopyridine (4-AP), an inhibitor of I_to, but had little effect on the subendocardial action potential. These data suggest that an increase in I_to may, at least in part, underlie the changes in action potential configuration recorded during simulated ischemia so that the regional differences in the response to such "ischemia" may reflect regional differences in the density of I_to.

Because acidosis is known to have marked effects on several membrane currents (for reviews see Refs. 23, 24) and is a major component of both the simulated ischemia described above and of true ischemia, then if acidosis increases I_to, this could explain the changes in action potential configuration observed by Antzelevitch et al. (3) and could play a role in altering the electrical activity of the heart during true ischemia. We have, therefore, investigated the effects of acidosis on I_to in cells isolated from the subepicardial and subendocardial regions of the rat left ventricle.

	MATERIALS AND METHODS

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Cell Isolation

1

1

Once the preparation appeared stable and the coronary vessels had cleared of blood, the perfusion was switched to nominally Ca²⁺-free PSS for 4 min. The perfusate was then switched to PSS containing 1 mg/ml collagenase (type II, Worthington), 0.1 mg/ml protease (type XIV, Sigma), and 50 µM Ca²⁺. This solution was recirculated to give a total exposure to the enzyme of 10 min.

At the end of the perfusion, the heart was cut down and the ventricles dissected free. The ventricles were then cut open, and thin slices of tissue were cut from the subepicardium (<2 mm from the epicardial surface) and the subendocardium (<2 mm from the endocardial surface) of the left ventricle using a fine pair of scissors. The slices were cut parallel to the surface of the ventricular wall and along the apex-base axis. The slices from each region were placed in separate conical flasks and gently agitated in enzyme-containing PSS supplemented with 1% BSA (Sigma) for 5-min periods at 37°C. Cells from each 5-min incubation were harvested by filtration followed by centrifugation at 400 rpm for 40 s. The supernatant was removed, and the cells were resuspended in PSS containing 0.75 mM Ca²⁺. This procedure was repeated four or five times for tissue from each region, and the cells stored until use.

Experimental Procedure

Perforated Patch Recording

Solutions and Drugs

Extracellular acidosis was produced by lowering the pH of the HPS bathing solution from 7.4 to 6.5. Reducing extracellular pH (pH_o) in a HPS reduces intracellular pH (pH_i) by 20-40% of the change of pH_o in ventricular muscle (6). Thus, in the present study, we would expect pH_i to decrease from its normal value of ~7.1 to between pH 6.9 and 6.7 in the presence of the acid solution. Previous work in our laboratory (18) has shown that pH_i decreases to a new steady state within 5-min exposure to this type of acidosis so that all of the measurements reported in the present paper were obtained after at least 5-min exposure to the acid solution. Nifedipine (10 µM) was always present in the bathing solution to block the L-type calcium current (I_Ca). 4-AP was prepared as a 100 mM stock solution (pH set to 7.4 using 1 M HCl) that was diluted to give a final concentration of 10 mM. Care was taken to adjust the pH of the experimental solution to the desired value (pH 7.4 or 6.5) after the addition of 4-AP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM was dissolved in DMSO to produce a 1 M stock solution that was added to the bathing solution to give a final concentration of 5 µM. This concentration of BAPTA-AM was sufficient to abolish contraction in field-stimulated myocytes in the absence of nifedipine (data not shown). We have previously shown (18) that BAPTA does not affect pH_i or its response to acidosis.

Experimental Protocol and Data Analysis

All data are expressed as means ± SE of n cells. Statistical comparisons were made using an unpaired or paired t-test as appropriate. Values of P < 0.05 were taken to indicate statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of Acidosis on I_to in Different Regions of the Rat Left Ventricle

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of acidosis on transient outward K+ current (Ito) in a subepicardial cell and subendocardial cell from a holding potential of 80 mV. A and B: voltage-clamp pulse protocol (top) and superimposed records of membrane current recorded from a holding potential of 80 mV at control pH [extracellular pH (pHo) 7.4; A] and during acidosis (pHo 6.5; B) in a representative subepicardial (Epi; left) and subendocardial cell (Endo; right). Membrane current was recorded during 200-ms depolarizing pulses to potentials between 0 and +60 mV. Arrows indicate the zero-current level, and the gaps in the current records indicate where current during prepulse to 40 mV (see MATERIALS AND METHODS) has been removed for clarity.

Figure 2 shows superimposed current records from a representative subepicardial and subendocardial cell, recorded from a holding potential of 40 mV at pH_o 7.4 (A) and 6.5 (B). At control pH, depolarizing pulses to potentials more positive than 20 mV activated little I_to in cells from either the subepicardium (Fig. 2A, left) or subendocardium (Fig. 2A, right); mean current amplitude at +60 mV in subepicardial and subendocardial cells was 0.13 ± 0.06 nA (n = 8) and 0.12 ± 0.05 nA (n = 4), respectively (P = NS). Membrane currents recorded from 40 mV in the same cells during acidosis are shown in Fig. 2B. After 5-min exposure to acidosis, depolarizing pulses to potentials positive to 20 mV resulted in the voltage-dependent activation of an outward current in subepicardial cells (Fig. 2B, left); peak outward current amplitude at +60 mV increased significantly (n = 8, P < 0.001) to 1.28 ± 0.12 nA, and the current remaining at the end of the depolarizing pulse increased from 0.35 ± 0.04 nA at +60 mV at control pH to 0.43 ± 0.05 nA during acidosis (n = 8; P < 0.05). In contrast, in subendocardial cells (Fig. 2B, right), outward current recorded during acidosis was not significantly different from that at control pH (Fig. 2A, right; n = 4). Acidosis-sensitive currents (obtained by subtracting membrane currents recorded at control pH from those obtained during acidosis) in a representative subepicardial and subendocardial cell are shown in Fig. 2C. In subepicardial cells (Fig. 2C, left), the acidosis-sensitive currents were outward currents that increased in amplitude as the voltage was stepped to more positive potentials and showed rapid activation and inactivation. In contrast, little acidosis-induced current was observed in subendocardial cells (Fig. 2C, right).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of acidosis on Ito in a subepicardial cell and a subendocardial cell from a holding potential of 40 mV. A and B: membrane potential (top) and superimposed records of membrane current recorded from a holding potential of 40 mV at control pH (pHo 7.4; A) and during acidosis (pHo 6.5; B) in a representative subepicardial (left) and subendocardial cell (right). Membrane current was recorded during 200-ms depolarizing pulses to potentials between 0 and +60 mV. C: superimposed acidosis-sensitive current in subepicardial cells (left) and subendocardial cells (right), obtained by subtracting current at control pH from the current during acidosis. Arrows indicate zero-current level.

Figure 3 shows mean (±SE, n = 8) data comparing the effects of acidosis on the current-voltage relationship of subepicardial cells at the two holding potentials. Current-voltage relationships were constructed by plotting current amplitude (measured as described in MATERIALS AND METHODS) against test potential. Figure 3A shows that I_to activated from a holding potential of 80 mV was large and not significantly altered by acidosis. However, at a holding potential of 40 mV (Fig. 3B), I_to was small at control pH, but acidosis significantly (P < 0.001) increased outward current at potentials more positive than 20 mV. This is in contrast to the data obtained in subendocardial cells, in which I_to was small at both holding potentials and was not significantly increased by acidosis.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of acidosis on current-voltage relationship of Ito at holding potentials of 80 and 40 mV in subepicardial cells. A and B: current-voltage relationship of Ito at control pHo () and during acidosis () at holding potentials of 80 mV (A) and 40 mV (B) in subepicardial cells. Ito (measured as difference between peak outward current and current at the end of the 200-ms depolarizing pulse) is plotted against test potential. Means ± SE are shown (n = 8).

Identity of the Acidosis-Sensitive Outward Current in Subepicardial Cells

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of 4-aminopyridine (4-AP) on acidosis-induced increase in Ito in subepicardial cells. A and B: superimposed records of membrane potential (top) and membrane current (bottom) recorded from a holding potential of 40 mV during acidosis before (A) and after (B) application of 10 mM 4-AP. Membrane current was recorded during 200-ms depolarizing pulses to potentials between 0 and +60 mV. C: superimposed 4-AP-sensitive currents, calculated by subtracting current after application of 4-AP from that before application of 4-AP during acidosis. Arrows indicate zero-current level. D: mean (±SE) current-voltage relationship of Ito at control pH () and during acidosis before () and after () application of 4-AP.

Mechanisms Underlying the Acidosis-Induced Outward Current in Subepicardial Cells

Voltage dependence of inactivation of I_to. One possible explanation for the observed effect of acidosis on I_to is that the inactivation of I_to is shifted to more positive potentials during acidosis. To test this possibility, the effect of acidosis on the voltage dependence of inactivation of I_to was investigated using a double-pulse protocol; a 500-ms conditioning prepulse from a holding potential of 80 mV to potentials between 80 and 0 mV (in 10-mV steps) was followed by a 160-ms depolarizing test pulse to +60 mV (Fig. 5). I_to was measured during the test pulse, and its magnitude was related to the potential during the conditioning pulse. Figure 5 shows superimposed current records obtained during the conditioning and test pulses at control pH (Fig. 5A) and during acidosis (Fig. 5B). Figure 5A shows that at control pH little inactivation of I_to during the test pulse was observed when the conditioning pulse potential was more negative than 60 mV, but that the magnitude of I_to during the test pulse decreased (i.e., inactivation increased) as the conditioning pulse potential became more positive. Acidosis had no significant effect on the magnitude of I_to during the test pulse when the conditioning prepulse was more negative than 60 mV but caused a shift in the voltage dependence of inactivation so that more I_to remained after a conditioning prepulse to, e.g., 40 mV (see Fig. 5B). This is shown more clearly in Fig. 5C, which shows the effect of acidosis on the mean (±SE) steady-state inactivation curve of I_to. I_to during each test pulse was normalized to maximal I_to (I/I_max) and plotted against the voltage during the conditioning prepulse. These data were fitted by the Boltzmann equation
where V_0.5 is the conditioning potential that gives I/I_max = 0.5, V_m is the conditioning potential, and k describes the steepness of the curve.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of acidosis on the steady-state inactivation of Ito in subepicardial cells. A and B: measurement of voltage dependence of inactivation. Top panels show protocol: 500-ms conditioning prepulses to various potentials (from 80 to 0 mV in 10-mV increments) were followed by a 160-ms test pulse to +60 mV. Holding potential was 80 mV. Bottom panels show superimposed current records during conditioning pulses and subsequent test pulses to +60 mV at control pH (A) and during acidosis (B). Arrows indicate zero-current level. C: mean (±SE; n = 8) steady-state inactivation curves of Ito. Ito during each test pulse was normalized to maximal Ito (I/Imax) and plotted against conditioning prepulse potential (Vconditioning). Data are fitted with a Boltzmann equation. See text for further details.

Ca²⁺ dependence of I_to. Because intracellular Ca²⁺ concentration ([Ca²⁺]_i) increases during acidosis (2), and may affect I_to (10, 26), the possible role of [Ca²⁺ ]_i in the observed response of I_to to acidosis was investigated using the Ca²⁺ chelator BAPTA-AM to buffer [Ca²⁺]_i. Figure 6 shows superimposed records of I_to during acidosis before (A) and after (B) 10-min exposure to 5 µM BAPTA-AM. Membrane currents were recorded during 200-ms depolarizing pulses to test potentials between 0 and +60 mV from a holding potential of 40 mV. The amplitude of I_to evoked from a holding potential of 40 mV during acidosis was not significantly altered by buffering [Ca²⁺]_i. I_to at +60 mV before and after application of BAPTA was 1.18 ± 0.17 and 1.22 ± 0.13 nA, respectively (n = 4). This suggests that an increase in cytoplasmic [Ca²⁺] does not underlie the increase in amplitude of I_to that occurs during acidosis.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of buffering intracellular Ca2+ by BAPTA-AM on membrane current during acidosis in subepicardial cells at a holding potential of 40 mV. A and B: superimposed records of membrane potential (top) and membrane current (bottom) recorded from same cell during acidosis before (A) and after (B) application of 5 µM BAPTA-AM. Membrane current was recorded during 200-ms depolarizing pulses to potentials between 0 and +60 mV (in 10-mV increments) from a holding potential of 40 mV. C: superimposed records of BAPTA-sensitive currents calculated by subtracting currents after application of BAPTA-AM from those during acidosis alone. Arrows indicate zero-current level.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identity of the Acidosis-Sensitive Outward Current in Subepicardial Cells

However, because acidosis increases cytoplasmic [Ca²⁺] (2) and alters Na⁺/Ca²⁺ exchange activity, another possibility is that changes in Na⁺/Ca²⁺ exchange current (I_Na/Ca) might contribute to the observed current. However, nifedipine, which was present in the bathing solution to inhibit I_Ca, also abolishes the Ca²⁺ transient and contraction so that there should be little contamination of I_to by Ca²⁺ transient-induced activation of I_Na/Ca. The idea that the amplitude of I_to was not contaminated by I_Na/Ca is supported by the observation that BAPTA had little effect on the amplitude of I_to during acidosis (Fig. 6). Thus it seems most likely that the outward current activated by acidosis from depolarized holding potentials is I_to.

Mechanism Underlying the Effect of Acidosis on I_to in Subepicardial Cells

A third possibility is that acidosis alters the voltage dependence of inactivation of I_to. In support of this hypothesis, acidosis caused a significant and reversible depolarizing shift in the steady-state inactivation of I_to (Fig. 5). At control pH in the present study, the half-maximal inactivation voltage and the slope of the inactivation curve were similar to those reported previously for I_to under similar experimental conditions (22), although more positive V_0.5 values (by ~13 mV) have also been reported (4, 9). This discrepancy may be related to the experimental conditions used to isolate I_to from other currents. In particular, divalent cations, which are frequently used to block I_Ca, shift the activation and inactivation curves to more positive potentials (1).

At control pH, when the conditioning prepulse potential was 80 mV, no inactivation of I_to was observed during the subsequent test pulse (Fig. 5). Because acidosis did not affect inactivation when the conditioning pulse was 80 mV (i.e., there was still no inactivation; Fig. 5), this could explain why acidosis had no effect on I_to when the holding potential was 80 mV (Fig. 3).

Changing the potential of the conditioning prepulse from 80 to 40 mV at control pH increased inactivation, thus reducing I_to during the subsequent test pulse by ~80%, which could explain why changing the holding potential from 80 to 40 mV at control pH decreased I_to, measured at +60 mV, by ~93% (cf. Fig. 3).

Acidosis produced a depolarizing shift of the inactivation curve of ~8.6 mV so that there was less inactivation after a conditioning prepulse to 40 mV. Thus, when the holding potential was 40 mV, significant inactivation of I_to would occur at control pH, but the acidosis-induced shift in the inactivation curve would decrease the proportion of inactivated current. After a conditioning prepulse to 40 mV during acidosis, I_to was ~75% of maximal I_to, which could explain why acidosis increased I_to, measured at +60 mV, to ~73% of maximal I_to when the holding potential was 40 mV (cf. Fig. 3). The rightward shift in the inactivation curve could, therefore, explain why acidosis increased I_to when the holding potential was 40 mV.

The mechanism(s) underlying the shift in the voltage-dependent inactivation of I_to is unknown, but it appears likely that it involves specific effects on the channel protein rather than changes of membrane surface charge, because inactivation, but not activation, was altered by acidosis. It is not clear, however, whether acidosis is acting at an intracellular or extracellular site. During the present experiments, the observed change in I_to was almost complete within 45 s of exposure to the acid solution (data not shown). We have previously shown that within this time pH_i has decreased little (by ~25% of its final change; Ref. 18), whereas with the bath volume (0.1 ml) and perfusion rate used (2.5 ml/min), the change of pH_o would be complete. Thus, although we cannot exclude an intracellular site of action, these data are compatible with an extracellular site being more important in the observed changes.

Ca²⁺ Dependence of I_to

Although buffering [Ca²⁺]_i during acidosis had no effect on the amplitude of I_to, inactivation was faster and the current remaining at the end of the pulse was decreased in the presence of BAPTA, suggesting that an acidosis-induced rise in [Ca²⁺]_i might underlie, at least in part, the acidosis-induced increase in the sustained outward current. The amplitude, time course, voltage dependence, and 4-AP insensitivity of the BAPTA-sensitive currents recorded during acidosis (Fig. 6C) were similar to the sustained outward current reported previously (4), although it remains possible that this sustained component is due to the activation of another Ca²⁺-activated current.

Regional Differences in the Response to Acidosis

Antzelevitch et al. (3) reported that simulated ischemia (6 mM K⁺, 95% N₂-5% CO₂; pH 6.8) caused a marked depression of the subepicardial action potential, which could be reversed by 4-AP, but had little effect on the subendocardial action potential. This suggests that an increase in I_to may play a role in the observed changes in action potential configuration and that the regional differences in the response to such "ischemia" may reflect regional differences in the density of I_to. Our data are consistent with this hypothesis and suggest that the acid component of the simulated ischemia may be important in the response to this intervention. Thus the increase in I_to that we observed during acidosis in subepicardial cells may explain the depression of the subepicardial action potential observed during simulated ischemia, and the lack of effect of acidosis on I_to in subendocardial cells may explain why the subendocardial action potential was little affected by simulated ischemia.

In contrast to the present data, Xu and Rozanski (31) reported that lowering pH_i decreases I_to in rat ventricular myocytes. However, acidosis and control data were obtained in different groups of cells. Because there is a marked heterogeneity in the amplitude of I_to in different regions of the heart and the authors did not separate epicardial and endocardial cells, they could not rule out the possibility that the observed difference could be due to the different cell types being predominant in each population (acid and control). In addition, these authors only observed a decrease when pH_i was lowered by dialysis with an acid (pH 6.0) pipette solution. When the pH of the bathing solution was lowered, as in the present study, acidosis did not significantly alter I_to when recorded from a holding potential of 80 mV, consistent with the results of the present study.

The present data show that acidosis increases I_to when the diastolic membrane potential is lower than the normal ventricular diastolic potential. This suggests that acidosis is most likely to increase I_to in regions of the heart where the diastolic potential is relatively positive, e.g., in the sinoatrial and atrioventricular nodes, or in pathological conditions, such as myocardial ischemia, in which an accumulation of extracellular potassium results in depolarization of the cell membrane. This suggests that either global acidosis (e.g., resulting from systemic acidosis) or regional acidosis (e.g., that found in the ischemic region during myocardial ischemia) may increase I_to in regions of the heart that, either physiologically or pathophysiologically, have a depolarized membrane and are exposed to the acidosis.

Thus the data from the present study suggest that acidosis will not increase I_to uniformly throughout the heart for a number of reasons: 1) because I_to is not uniformly distributed throughout the heart; 2) because diastolic membrane potential, which influences the response of I_to to acidosis, is not uniform throughout the heart; and 3) some types of acidosis found in vivo, e.g., that associated with myocardial ischemia, are localized within the heart. If acidosis does not increase I_to uniformly throughout the heart, this will alter action potential configuration differently in different regions of the heart, thus altering action potential dispersion, which can alter the normal spread of the action potential and may lead to arrhythmias. Thus it appears that the acidosis-induced increase in I_to observed in the present study may have marked effects on the normal electrical activity of the intact heart.