The effect of acidosis on the transient outward K+ current (Ito ) of rat ventricular myocytes has been investigated using the perforated patch-clamp technique. When the holding potential was −80 mV, depolarizing pulses to potentials positive to −20 mV activated Ito in subepicardial cells but activated little Ito in subendocardial cells. Exposure to an acid solution (pH 6.5) had no significant effect on Ito activated from this holding potential in either subepicardial or subendocardial cells. When the holding potential was −40 mV, acidosis significantly increased Ito at potentials positive to −20 mV in subepicardial cells but had little effect onIto in subendocardial cells. The increase inIto in subepicardial cells was inhibited by 10 mM 4-aminopyridine. In subepicardial cells, acidosis caused a +8.57-mV shift in the steady-state inactivation curve. It is concluded that in subepicardial rat ventricular myocytes acidosis increases the amplitude of Ito as a consequence of a depolarizing shift in the voltage dependence of inactivation.
- cardiac electrophysiology
the transient outward K+ current (Ito ) 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 Ito . Thus the action potential has a characteristic “spike and dome” morphology in the subepicardium, where Ito is prominent, that is less marked in the subendocardium, whereIto 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 ofIto in different regions of the heart, interventions that modulate Ito 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 alterIto , 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 Ito , but had little effect on the subendocardial action potential. These data suggest that an increase in Ito 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 Ito .
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 Ito , 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 Ito in cells isolated from the subepicardial and subendocardial regions of the rat left ventricle.
MATERIALS AND METHODS
Rat ventricular myocytes were isolated as described previously (13). Briefly, adult Wistar rats of either sex were stunned and then killed by cervical dislocation. The heart was rapidly removed and transferred to a beaker containing oxygenated physiological salt solution (PSS; for composition see below) where it was gently massaged to remove excess blood. The aorta was cannulated, and the heart was retrogradely perfused at a constant flow (8 ml ⋅ min−1 ⋅ g wet wt−1) with PSS containing Ca2+ (0.75 mM) at 37°C.
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.
When required, a drop of cell suspension containing subepicardial or subendocardial cells was placed in a small chamber (volume 0.1 ml) on the stage of a Nikon Diaphot inverted microscope. After 10–15 min, when the cells had settled onto the glass bottom of the chamber, the chamber was perfused at ∼2.5 ml/min with a HEPES-buffered PSS (HPS; for composition see below) at room temperature (22°C). Miniature solenoid valves (Lee Products) were used to direct one of up to four different solutions to the chamber. Solution was removed continuously to waste using suction.
Perforated Patch Recording
Membrane current was recorded using the perforated patch-clamp technique. Briefly, glass pipettes were pulled from nonheparinized hematocrit tubes to a resistance of 2–4 MΩ. The tip of the pipette was first filled with an antibiotic-free intracellular solution (see below for composition) that contained 1 mM Ca2+, to detect accidental rupture of the patch membrane (i.e., whole cell configuration), in which case the cell died rapidly; the pipette was then backfilled with the same solution plus 0.24 mM amphoteracin B. After correction of the liquid juction potential and gigaohm seal formation, 20-ms voltage command pulses from −40 to 0 mV were applied to the pipette using an Axopatch ID (Axon Instuments) patch-clamp amplifier. Electrical access was usually obtained within 5–10 min, and 20–30 min were allowed before starting the experiment. Pipette capacitance was electronically compensated, and membrane current was filtered at 1.5 kHz (low-pass Bessel filter). Signals were displayed on an oscilloscope (Tektronix 5111A) and pen recorder (Gould, RS3600). The signals were also recorded onto videotape via a pulse-code modulator (Neuro-Corder, Neuro Data Instruments, and JVC HR-D367 video recorder) and computer via a CED 1401 analog-to-digital converter (Cambridge Electronic Design) for later off-line analysis.
Solutions and Drugs
The PSS used during the cell isolation contained (in mM) 130 NaCl, 5.4 KCl, 0.4 Na2PO4, 1.4 MgCl2 ⋅ 6H2O, 10 HEPES, 10 glucose, 20 taurine, 10 creatine, 0.75 Ca2+, with pH set to 7.3 using 2 M NaOH. The enzyme-containing solution consisted of isolation solution plus 1 mg/ml collagenase (type I, Worthington), 0.1 mg/ml protease (type XIV, Sigma), and 50 μM CaCl2. The HPS used during the experiments contained (in mM) 130 NaCl, 5 KCl, 1 Na2HPO4 ⋅ 12H2O, 1 MgSO4 ⋅ 7H2O, 20 sodium acetate, 10 glucose, 5 HEPES, 1 CaCl2, and 5 U/l insulin, pH 7.4. The pipette solution contained (in mM) 110 potassium glutamate, 10 KCl, 10 NaCl, 1 MgCl2, 5 HEPES, and 1 CaCl2, pH 7.1.
Extracellular acidosis was produced by lowering the pH of the HPS bathing solution from 7.4 to 6.5. Reducing extracellular pH (pHo) in a HPS reduces intracellular pH (pHi) by 20–40% of the change of pHo in ventricular muscle (6). Thus, in the present study, we would expect pHi 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 pHi 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 pHi or its response to acidosis.
Experimental Protocol and Data Analysis
The holding potential was set to either −80 or −40 mV, as described in results, and voltage-clamp pulses were applied at 0.5 Hz. When the holding potential was −80 mV, 50-ms depolarizing pulses to −40 mV were applied (to inactivate the Na+ current) immediately before the 200-ms depolarizing test pulses to various potentials that were used to activateIto . The magnitude of the transient outward component of Ito during each test pulse was measured as the difference between peak outward current and the current remaining at the end of the 200-ms depolarizing pulse. The magnitude of the sustained outward current was measured as the difference between the current remaining at the end of the 200-ms depolarizing pulse and the holding current between pulses. Acidosis-sensitive, 4-AP-sensitive, and BAPTA-sensitive currents are presented as the difference between the currents measured in the presence of acidosis, 4-AP, or BAPTA and those measured in their absence.
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.
Effect of Acidosis on Ito in Different Regions of the Rat Left Ventricle
Figure 1 shows superimposed records ofIto in a representative subepicardial and subendocardial cell, recorded from a holding potential of −80 mV at control pH (pHo 7.4; Fig. 1 A) and after 5-min exposure to the acid solution (pHo 6.5; Fig. 1 B). At control pH, 200-ms depolarizing pulses to potentials more positive than −20 mV resulted in the voltage-dependent activation ofIto in cells isolated from the subepicardium (Fig.1 A, left). In contrast, little Ito was recorded in cells isolated from the subendocardium (Fig.1 A, right). In eight subepicardial cells, mean current amplitude at +60 mV (measured as described in materials and methods) was 1.79 ± 0.35 nA, whereas current amplitude at +60 mV in four subendocardial cells was 0.14 ± 0.09 nA (P < 0.001). Currents recorded from the same subepicardial and subendocardial cells during acidosis are shown in Fig. 1 B. Five-minute exposure to the acid solution did not significantly alterIto in either subepicardial or subendocardial cells; mean current amplitude during acidosis at +60 mV in subepicardial and subendocardial cells was 1.81 ± 0.33 nA (n= 8) and 0.15 ± 0.07 nA (n = 4), respectively.
Figure 2 shows superimposed current records from a representative subepicardial and subendocardial cell, recorded from a holding potential of −40 mV at pHo 7.4 (A) and 6.5 (B). At control pH, depolarizing pulses to potentials more positive than −20 mV activated littleIto in cells from either the subepicardium (Fig.2 A, left) or subendocardium (Fig. 2 A,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. 2 B. 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. 2 B, 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. 2 B, right), outward current recorded during acidosis was not significantly different from that at control pH (Fig. 2 A,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. 2 C. In subepicardial cells (Fig. 2 C, 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. 2 C,right).
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 3 A shows thatIto 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. 3 B),Ito 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 whichIto was small at both holding potentials and was not significantly increased by acidosis.
Identity of the Acidosis-Sensitive Outward Current in Subepicardial Cells
To determine whether the acidosis-sensitive outward current recorded in subepicardial cells was Ito , the effect of 10 mM 4-AP, an inhibitor of Ito , was investigated. All effects of 4-AP shown were obtained in subepicardial cells, when the holding potential was −40 mV, and were recorded after 3-min exposure to 4-AP. Figure 4 shows the effect of 10 mM 4-AP on membrane currents obtained in response to 200-ms depolarizing pulses to different test potentials during acidosis. During acidosis in the absence of 4-AP, depolarizing pulses to potentials positive to −20 mV resulted in the rapid voltage-dependent activation of an outward current, which declined during the 200-ms pulse (Fig.4 A), as described above. Application of 4-AP markedly reduced peak outward current, with little effect on the sustained component of membrane current (Fig. 4 B). 4-AP-sensitive currents, obtained by subtracting current in the presence of 4-AP (Fig. 4 B) from that in its absence (Fig. 4 A), are shown in Fig. 4 C. The amplitude of the 4-AP-sensitive currents was voltage dependent; these currents also showed rapid activation and inactivation, with almost complete decay by the end of the 200-ms depolarizing pulse. Figure 4 D shows mean (±SE) current-voltage relationships at control pH, during acidosis, and during acidosis in the presence of 4-AP, showing that 4-AP significantly inhibited the acidosis-induced outward current amplitude at potentials positive to −20 mV (n = 5; P < 0.01) so that the current-voltage relationship in the presence of 4-AP was not significantly different from that observed at control pH. These effects of 4-AP were reversed when 4-AP was removed from the perfusate (data not shown). These data suggest, therefore, that the outward current activated by acidosis is Ito (seediscussion).
Mechanisms Underlying the Acidosis-Induced Outward Current in Subepicardial Cells
Voltage dependence of inactivation of Ito.
One possible explanation for the observed effect of acidosis onIto is that the inactivation ofIto is shifted to more positive potentials during acidosis. To test this possibility, the effect of acidosis on the voltage dependence of inactivation of Ito 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).Ito 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. 5 A) and during acidosis (Fig. 5 B). Figure 5 A shows that at control pH little inactivation of Ito during the test pulse was observed when the conditioning pulse potential was more negative than −60 mV, but that the magnitude of Ito 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 Ito 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 Ito remained after a conditioning prepulse to, e.g., −40 mV (see Fig. 5 B). This is shown more clearly in Fig. 5 C, which shows the effect of acidosis on the mean (±SE) steady-state inactivation curve ofIto. Ito during each test pulse was normalized to maximal Ito (I/I max) and plotted against the voltage during the conditioning prepulse. These data were fitted by the Boltzmann equation whereV 0.5 is the conditioning potential that givesI/I max = 0.5,V m is the conditioning potential, and kdescribes the steepness of the curve.
Figure 5 C shows that at control pH there was little inactivation of Ito during the test pulse when the conditioning prepulses were more negative than −60 mV, but inactivation increased as the conditioning prepulse became more positive, and Ito was almost fully inactivated by conditioning prepulses positive to −30 mV. The values ofV 0.5 and k at pHo 7.4 were −45.97 mV and 3.98 mV, respectively (n = 8). Figure5 C also shows that acidosis significantly (P < 0.05) shifted the inactivation curve to more positive potentials (with no significant effect on the slope of the relationship) so that after conditioning prepulses to potentials between −50 and −30 mV, there was less inactivation during acidosis than at control pH. Acidosis caused an 8.57-mV depolarizing shift in the inactivation curve; V 0.5 at pHo 6.5 was −37.4 mV, and k was 3.87 mV (n = 8). These data are compatible, therefore, with the hypothesis that the effects of acidosis on the amplitude of Ito are due to its effects on the inactivation of Ito (seediscussion).
Ca2+ dependence of Ito.
Because intracellular Ca2+ concentration ([Ca2+]i) increases during acidosis (2), and may affect Ito (10, 26), the possible role of [Ca2+ ]i in the observed response of Ito to acidosis was investigated using the Ca2+ chelator BAPTA-AM to buffer [Ca2+]i. Figure6 shows superimposed records ofIto 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 ofIto evoked from a holding potential of −40 mV during acidosis was not significantly altered by buffering [Ca2+]i. Ito 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 Ito that occurs during acidosis.
Figure 6 also shows that the time course of inactivation was more rapid in the presence of BAPTA. The decay of Ito at +60 mV was fitted using a double exponential function that gave time constants (τ) for the fast phase of inactivation during acidosis of 36.3 ± 2.6 ms before, and 23.7 ± 1.6 ms after, the application of BAPTA-AM (P < 0.05; n = 4). However, the time constants for the slow phase of inactivation during acidosis before and after application of BAPTA-AM were not significantly different (1,523 ± 387 and 1,160 ± 213 ms, respectively). In addition, BAPTA reduced the current remaining at the end of the depolarizing pulse (Fig. 6) from 0.38 ± 0.07 nA in the absence of BAPTA to 0.27 ± 0.08 nA in the presence of BAPTA (n = 4, P < 0.05). Figure 6 C shows the BAPTA-sensitive currents, which were obtained by subtracting the currents obtained during acidosis in the presence of BAPTA from those obtained in the presence of acidosis alone; these are sustained outward currents that show rapid activation on depolarization and decay slowly to an apparent plateau.
Identity of the Acidosis-Sensitive Outward Current in Subepicardial Cells
When the holding potential was −40 mV, acidosis activated a rapidly activating and inactivating outward current in subepicardial cells. It appears likely that this current was Ito for several reasons: 1) it was blocked when K+ in the pipette solution was replaced by Cs+ (data not shown), suggesting that this current is carried predominantly by K+; 2) it was inhibited by 4-AP, an inhibitor of the Ca2+-independent component of Ito ;3) the 4-AP-sensitive and the acidosis-sensitive currents were similar, both exhibiting an amplitude, time course, and current-voltage relation characteristic of Ito ; and 4) the observation that the effects of acidosis were greater in subepicardial cells than subendocardial cells is consistent with the previously reported distribution of Ito . These data suggest that the outward current activated by acidosis from depolarized potentials is Ito , consistent with a recent study by Stengl et al. (27).
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 Ito by Ca2+ transient-induced activation of I Na/Ca. The idea that the amplitude of Ito was not contaminated byI Na/Ca is supported by the observation that BAPTA had little effect on the amplitude of Ito during acidosis (Fig. 6). Thus it seems most likely that the outward current activated by acidosis from depolarized holding potentials isIto .
Mechanism Underlying the Effect of Acidosis on Ito in Subepicardial Cells
Although acidosis increased Ito in subepicardial cells, the effect was strongly voltage dependent; acidosis increasedIto when the holding potential was −40 mV but had little effect on Ito when the holding potential was −80 mV. This voltage dependence could be due to several possible mechanisms. One possibility is that acidosis alters the rate of recovery of Ito from inactivation. It has previously been shown (30) that recovery of Ito is voltage dependent, being slower at more depolarized potentials. However, even at −40 mV, recovery is complete within 2 s (the interpulse interval used in the present study, Ref. 30). Thus if acidosis slowed recovery, it would be expected to decreaseIto rather than increase it as seen in the present study; if acidosis accelerated recovery, it would be expected to have little or no effect on the magnitude of Ito . It appears unlikely, therefore, that acidosis-induced changes in recovery can explain the present data. A second possibility is that the threshold for activation of Ito could be shifted to more negative potentials during acidosis, since protons may screen fixed negative charges on the membrane, thus altering the potential detected by the channel (16). However, this seems unlikely, because acidosis did not produce a leftward shift of the current-voltage relationship (Fig. 3).
A third possibility is that acidosis alters the voltage dependence of inactivation of Ito . In support of this hypothesis, acidosis caused a significant and reversible depolarizing shift in the steady-state inactivation of Ito (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 Ito 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 isolateIto 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 Ito 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 Ito 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 reducingIto during the subsequent test pulse by ∼80%, which could explain why changing the holding potential from −80 to −40 mV at control pH decreased Ito , 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 Ito 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,Ito was ∼75% of maximal Ito , which could explain why acidosis increased Ito , measured at +60 mV, to ∼73% of maximal Ito when the holding potential was −40 mV (cf. Fig. 3). The rightward shift in the inactivation curve could, therefore, explain why acidosis increased Ito when the holding potential was −40 mV.
The mechanism(s) underlying the shift in the voltage-dependent inactivation of Ito 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 Ito was almost complete within 45 s of exposure to the acid solution (data not shown). We have previously shown that within this time pHi 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 pHo 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 Ito
The existence of both Ca2+-dependent and -independent components of Ito has been reported in several studies (10, 12, 14, 17, 28), whereas in others, the presence of a single Ca2+-independent component ofIto has been described (4, 7, 11, 15, 19). In the present study, buffering [Ca2+]iwith BAPTA had no effect on the amplitude of Ito (Fig. 6), suggesting that the increase in [Ca2+]i that occurs during acidosis does not underlie the increase in Ito observed in the present study. It is, however, worth noting that in the present study the Ca2+ transient was inhibited by nifedipine (so that Ito could be monitored, see materials and methods) before the application of BAPTA-AM. Thus, in the present study, effects mediated by the Ca2+ transient would not be observed, and any changes observed (below) are likely to be due to the effects of acidosis on diastolic, rather than systolic, [Ca2+]i.
Although buffering [Ca2+]i during acidosis had no effect on the amplitude of Ito , 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+]imight 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. 6 C) 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
Recent studies have shown marked heterogeneity in the electrophysiological properties of the epicardium and endocardium of the ventricle in several species, including the rat (29). Differences in action potential configuration in the subepicardial and subendocardial regions of the ventricle have been attributed to the prominence of Ito in the subepicardium and its relative absence in the subendocardium (21; also see the introduction). Consistent with this view, we found that Ito was more marked in subepicardial cells than in subendocardial cells (Fig.1) and that acidosis increased Ito in subepicardial, but not in subendocardial, cells when the holding potential was −40 mV; the greater effects of acidosis in the subepicardium may be due to the greater density ofIto in subepicardial cells.
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 Ito 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 ofIto . 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 Ito 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 Ito 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 pHi decreases Ito 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 Ito 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 pHi 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 Ito when recorded from a holding potential of −80 mV, consistent with the results of the present study.
The present data show that acidosis increases Ito when the diastolic membrane potential is lower than the normal ventricular diastolic potential. This suggests that acidosis is most likely to increase Ito 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 increaseIto 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 Ito uniformly throughout the heart for a number of reasons: 1) because Ito is not uniformly distributed throughout the heart; 2) because diastolic membrane potential, which influences the response ofIto 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 Ito 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 Ito observed in the present study may have marked effects on the normal electrical activity of the intact heart.
We thank the British Heart Foundation for financial support and Professor M. R. Boyett for helpful assistance and discussion.
Address for reprint requests and other correspondence: C. H. Orchard, School of Biomedical Sciences, Univ. of Leeds, Leeds LS2 9JT, UK (E-mail:).
J. T. Hulme is a University of Leeds Research Scholar.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- Copyright © 2000 the American Physiological Society