Modulation of ventricular transient outward K⁺ current by acidosis and its effects on excitation-contraction coupling

Abstract

The contribution of transient outward current (I_to) to changes in ventricular action potential (AP) repolarization induced by acidosis is unresolved, as is the indirect effect of these changes on calcium handling. To address this issue we measured intracellular pH (pH_i), I_to, L-type calcium current (I_Ca,L), and calcium transients (CaTs) in rabbit ventricular myocytes. Intracellular acidosis [pH_i 6.75 with extracellular pH (pH_o) 7.4] reduced I_to by ∼50% in myocytes with both high (epicardial) and low (papillary muscle) I_to densities, with little effect on steady-state inactivation and activation. Of the two candidate α-subunits underlying I_to, human (h)Kv4.3 and hKv1.4, only hKv4.3 current was reduced by intracellular acidosis. Extracellular acidosis (pH_o 6.5) shifted I_to inactivation toward less negative potentials but had negligible effect on peak current at +60 mV when initiated from −80 mV. The effects of low pH_i-induced inhibition of I_to on AP repolarization were much greater in epicardial than papillary muscle myocytes and included slowing of phase 1, attenuation of the notch, and elevation of the plateau. Low pH_i increased AP duration in both cell types, with the greatest lengthening occurring in epicardial myocytes. The changes in epicardial AP repolarization induced by intracellular acidosis reduced peak I_Ca,L, increased net calcium influx via I_Ca,L, and increased CaT amplitude. In summary, in contrast to low pH_o, intracellular acidosis has a marked inhibitory effect on ventricular I_to, perhaps mediated by Kv4.3. By altering the trajectory of the AP repolarization, low pH_i has a significant indirect effect on calcium handling, especially evident in epicardial cells.

transient outward k⁺ current (I_to) is the major repolarizing current flowing during the early repolarization phase of the cardiac action potential (AP) in a variety of cell types including human and rabbit ventricular myocytes and is largely responsible for phase 1 and the “spike-and-dome” configuration (4, 12, 17, 20, 22, 31, 48). By modulating the initial voltage trajectory of the AP, I_to importantly influences the overall AP waveform through voltage-dependent effects on other ionic currents such as L-type calcium current (I_Ca,L), Na⁺/Ca²⁺ exchange (NCX) current, and delayed rectifier currents (34). In this regard, several studies have shown that inhibition of I_to markedly affects excitation-contraction (E-C) coupling by altering transsarcolemmal Ca²⁺ flux via I_Ca,L and NCX, with secondary effects on calcium loading of the sarcoplasmic reticulum (SR) (5, 6, 11, 13, 27, 42, 43, 53).

The expression and magnitude of cardiac I_to are regulated by a variety of factors and conditions including hormones, metabolic inhibition, and hypertrophy/failure with consequent effects on AP configuration (32, 36). In addition, previous work has shown that I_to in rat ventricular myocytes, measured at room temperature, is altered by extracellular acidosis [low extracellular pH (pH_o)] (50), intracellular acidosis [low intracellular pH (pH_i)] (58, 59), and simultaneous reductions in pH_o and pH_i (combined acidosis) (24). Low pH_o elicits right shifts in inactivation (toward less negative potentials) causing increased I_to at voltages of approximately +50 mV to +60 mV when initiated from depolarized potentials but has little or no effect when initiated from voltages near the normal resting potential (50). In contrast, intracellular acidosis (pH_o 7.4), induced by pipette acid loading, is reported to decrease I_to over the voltage range of −30 mV to +60 mV (58, 59). However, pH_i was not measured in these reports, and subsequent work has demonstrated that accurate pH_i dialysis via a suction pipette is very difficult to achieve, given the high intracellular H⁺ buffering and low H⁺ mobility (52, 61). Thus the quantitative relationship between ventricular pH_i and I_to remains unresolved. To address this issue, in the present study we measured pH_i and I_to at 37°C in rabbit ventricular myocytes, a preparation with a more humanlike AP than rat ventricle, using a more reliable technique for selectively reducing pH_i.

Also unresolved is the role of I_to in mediating pH-induced changes in AP repolarization and calcium handling. Using a mixture of rabbit myocytes from the entire left ventricle, we recently reported that a selective reduction in pH_i (pH_o 7.4) slowed the rate of phase 1 repolarization, reduced the notch, elevated the AP plateau, and prolonged AP duration (APD). These AP changes are consistent with I_to inhibition and may indirectly contribute to the effects of low pH_i on ventricular E-C coupling. However, because I_to exhibits a large transmural density gradient (epicardial > endocardial) (4, 9, 17, 18, 31), our use of a mixture of cells from the entire left ventricle meant that phase 1 and the notch varied considerably from one cell to another. Thus it remains unclear how pH_i-induced changes in I_to modulate the AP, I_Ca,L, and the calcium transient (CaT). Here we address this question in rabbit ventricular myocytes displaying both high [epicardial (Epi)] and low [papillary muscle (PM)] I_to densities, using AP voltage clamps and fluo-4 fluorescence.

Collectively the results demonstrate that intracellular acidosis markedly reduces ventricular I_to, which significantly contributes to the accompanying changes in early AP repolarization, resulting in indirect modulation of E-C coupling.

MATERIALS AND METHODS

Myocyte Isolation

The experiments were performed on left ventricular myocytes isolated from adult rabbits of both sexes by enzymatic digestion. To minimize transmural differences in I_Ca,L density (37), only male rabbits were used for the measurements of I_Ca,L and CaTs. All procedures involving animals were approved by the Animal Care and Use Committee of the University of Utah and complied with the American Physiological Society's “Guiding Principles for the Care and Use of Vertebrate Animals in Research and Training.” Rabbits were anesthetized with pentobarbital sodium (50 mg/kg iv), and the excised heart was attached to an aortic cannula and perfused with solutions gassed with 100% O₂ and held at 37°C, pH 7.3. The heart was first perfused for 5 min with a Ca²⁺-free solution at 37°C containing (in mM) 92.0 NaCl, 4.4 KCl, 11.0 dextrose, 5.0 MgCl₂, 20.0 taurine, 5.0 creatine, 5.0 sodium pyruvate, 1.0 NaH₂PO₄, 24.0 HEPES, and 12.5 NaOH (pH 7.3). This was followed by 15 min of recirculation with the same solution containing 0.15 mg/ml collagenase P (Roche Diagnostic, Mannheim, Germany), 0.05 mg/ml protease (type XIV, Sigma Chemical), and 0.05 mM CaCl₂. The heart was then perfused for 5 min with the same solution containing no enzymes. The ventricle was isolated and minced, shaken for 10 min, and then filtered through a nylon mesh. Cells were stored at room temperature in the normal control bathing solution. To obtain cells with both low and high I_to densities, myocytes were isolated from both PMs and the epicardial layers (Epi) of the left ventricle, respectively (17). All cells used in this study were rectangular, had well-defined striations, and did not spontaneously contract. All experiments were conducted within 10 h of isolation.

Cell Culture

Human embryonic kidney cells (HEK 293T) were cultured in DMEM supplemented with 10% fetal bovine serum, l-glutamine (2 mM), penicillin (100 IU/ml), and streptomycin (100 μg/ml). HEK 293T cells were plated onto 35-mm culture dishes and transfected with human (h)Kv1.4 (pcDNA3) along with green fluorescent protein (pEGFP) or hKv4.3 (pIRES-GFP) with Lipofectamine (Invitrogen). Experiments were conducted 1–2 days after transfection.

Cell Superfusion Chamber

Isolated ventricular cells were superfused at 4 ml/min (37 ± 0.3°C), solution exchange within the experimental chamber occurring in ∼5 s. The glass bottom of the chamber was coated with laminin (Collaborative Research, Bedford, MA) to improve cell adhesion. HEK 293T cells were bathed in solution at 23 ± 1.0°C without laminin coating the bottom of the bath.

Bathing Solutions and Drugs

Two types of acidosis were applied to myocytes: extracellular acidosis (pH_o 6.5, pH_i ∼7.1) and intracellular acidosis (pH_o 7.4, pH_i ∼6.7) as previously described (41). The normal control solution for extracellular acidosis (low pH_o) and intracellular acidosis (low pH_i) contained (mM) 126.0 NaCl, 11.0 dextrose, 4.4 KCl, 1.0 MgCl₂, 1.08 CaCl₂, and 24.0 HEPES titrated to pH 7.4 with 1 M NaOH. During acidosis experiments all bathing solutions contained 30 μM cariporide (Sanofi-Aventis, Frankfurt, Germany) to selectively block sodium/hydrogen exchange (NHE) and no added CO₂ or HCO₃⁻ to block transsarcolemmal transport of acid equivalents via Na⁺-HCO₃⁻ cotransport (NBC) (60) and Cl⁻/HCO₃⁻ exchange (57). The solution used to create extracellular acidosis had the same composition as the control solution except that its pH was titrated to 6.5 with NaOH. In separate experiments we found that 30 μM cariporide had no effect on I_to (n = 5, data not shown) or I_Ca,L (41).

The solution used to induce intracellular acidosis with pH_o held at 7.4 was prepared by equimolar replacement of 80.0 mM sodium acetate (NaAc) in the control solution for NaCl, as recently described (41). Decreasing pH_i by application of extracellular acetate is a widely used technique and results from rapid influx of uncharged protonated acetate, which then releases protons intracellularly. To compensate for the decrease in Ca²⁺ activity in the bathing solution due to acetate binding, CaCl₂ was increased in the 80 mM acetate solution from 1.08 mM to 1.37 mM (41). The bathing solutions for HEK cells had the same composition as those for myocytes except that 40 mM NaAc was used to induce intracellular acidosis.

During several voltage-clamp protocols, I_Ca,L and the inward-rectifying potassium current (I_K1) were blocked by CdCl₂ (200 μM; Sigma-Aldrich) and BaCl₂ (200 μM; Sigma-Aldrich), respectively. Sodium current (I_Na) was inhibited in some experiments by equimolar replacement of NaCl with N-methyl-d-glucamine, and pH_o was adjusted to 7.4 with HCl. In this case CaCl₂ was reduced to 0.1 mM to retard calcium overload upon exposure to sodium-free solution. I_to and the rapid delayed-rectifier potassium current (I_Kr) were inhibited in some experiments by including 3 mM 4-aminopyridine (Sigma-Aldrich) and 2 μM E-4031 (Sigma-Aldrich), respectively. In contrast to a previous report (47), BaCl₂ (200 μM) had no effect on I_to (n = 4, data not shown). To measure I_Ca,L, potassium chloride was replaced with 4.4 mM CsCl in the control solutions. A mixture of 10 μM nifedipine (Sigma-Aldrich) and 200 μM CdCl₂ was used to block I_Ca,L for I_Ca,L voltage-clamp experiments.

Pipette Filling Solutions

The normal filling solution used for recording APs in myocytes contained (in mM) 110.0 K-gluconate, 10.0 KCl, 5.0 Na-glucuronate, 5.0 MgATP, 5.0 phosphocreatine, 1.0 NaGTP, and 10.0 HEPES titrated to pH 7.2 with 1 M KOH. This solution was also used when recording CaTs that were initiated with AP voltage clamps (AP clamp), without simultaneous measurement of I_Ca,L. The filling solution used to measure I_to in myocytes and HEK 293T cells expressing Kv4.3 and Kv1.4 had the same composition as the normal filling solution except that it also contained 5.0 mM BAPTA. Corrections were made for liquid junction potentials. Buffering intracellular calcium with BAPTA eliminates Ca²⁺-activated Cl⁻ current, also known as I_to2, which is present in rabbit ventricular myocytes (22) and activated by extracellular acidosis (23). Intracellular BAPTA also blocks the CaT and thus minimizes the complicating effects of Ca²⁺-activated NCX (7). BAPTA is preferable to EGTA because of its faster Ca²⁺ binding kinetics and lower pH sensitivity (51).

The pipette filling solution used for I_Ca,L voltage-clamp experiments (rectangular voltage steps and AP clamps) contained (in mM) 120.0 CsCl, 5.0 NaCl, 10.0 tetraethylammonium chloride (TEA-Cl), 5.0 MgATP, 5.0 phosphocreatine, 1.0 NaGTP, 5.0 BAPTA, and 10.0 HEPES titrated to pH 7.2 with 1 M CsOH. Corrections were made for liquid junction potentials. BAPTA was not present in experiments in which I_Ca,L and CaTs were simultaneously recorded (see Table 2) and when only CaTs were recorded during AP clamps (see Fig. 12).

Table 2.

Effect of AP repolarization on simultaneously measured I_Ca,L and CaTs

	Epi Template (n = 5)		PM Template (n = 5)
	Control	Intracellular acidosis	Control	Intracellular acidosis
Initial peak ICa,L, pA/pF	−7.64 ± 0.59	−5.35 ± 0.70**	−4.24 ± 0.14##	−3.91 ± 0.14*
Net Ca2+ influx via ICa,L, pC/pF	0.31 ± 0.03	0.40 ± 0.05*	0.23 ± 0.02	0.27 ± 0.03*
CaT amplitude (F/F0)	1.95 ± 0.23	2.09 ± 0.23**	1.53 ± 0.07	1.55 ± 0.06
Rate of rise, F/F0/s	51.8 ± 16.4	65.2 ± 14.4*	32.4 ± 6.55	33.3 ± 2.49
E-C coupling gain, (F/F0/s)/(pA/pF)	7.36 ± 2.66	13.8 ± 4.22*	7.69 ± 1.60	8.58 ± 0.79

All values are means ± SE. All experiments were performed in the absence of acidosis with appropriate action potential (AP) templates for control and intracellular acidosis. I_Ca,L, L-type calcium current; CaT, calcium transient; Epi, epicardial; PM, papillary muscle; F/F₀, ratio of fluorescence during CaT divided by diastolic fluorescence; E-C, excitation-contraction.

^*P < 0.05,

^**P < 0.01 vs. control, paired; ^##P < 0.01 vs. control with Epi template, unpaired.

Fig. 12.

Response of calcium transients (CaTs) in Epi and PM myocytes to changes in AP repolarization induced by intracellular acidosis (80.0 mM acetate). Control bathing solution was used for all experiments shown in this figure. A: examples of CaTs elicited by AP clamps using control and acidosis templates in Epi (a) and PM (b) myocytes. B: summary of results for amplitude (a), maximum rate of rise (b), and duration (c) of CaTs (Epi, n = 8; PM, n = 10). The acidosis template increased amplitude and maximum rate of rise of the CaT in Epi but not PM cells. The acidosis template increased CaT duration [measured as time from upstroke to 50% recovery (CaTD₅₀)] in both cell types. *P < 0.05, **P < 0.01, paired, control vs. acidosis template. CL = 2 s was used for all experiments shown in this figure.

Intracellular acidosis (pH_o 7.4) was induced in some experiments with pipette acid loading. The filling solution contained (mM) 110.0 KCl, 5.0 NaCl, 5.0 MgATP, 5.0 phosphocreatine, 1.0 NaGTP, and 10.0 HEPES titrated to pH 6.6 with 1 M KOH.

Electrophysiological Techniques

All electrophysiological measurements in both myocytes and HEK 293T cells were made with whole cell ruptured patch pipettes. Pipettes (Corning 8250 glass) for myocytes and HEK 293T cells had resistances of 1–2 MΩ and 2.5–4.0 MΩ, respectively, when filled. Myocyte APs were recorded with an Axoclamp-2A amplifier system (Axon Instruments) in bridge mode, and voltage clamping (step clamps and AP clamps) was achieved with an Axopatch 200B clamp system using a CV203BU headstage. APs were triggered with brief (2–3 ms) square pulses of depolarizing intracellular current (∼2 nA), and APD was measured at 90% repolarization (APD₉₀). Membrane potential (V_m) and membrane current (I_to and I_Ca,L) were filtered at 5 kHz, digitized at 50 kHz with a 16-bit A/D converter (Digidata 1322A), and analyzed with pCLAMP 8 software (Molecular Devices). The reference electrode was a flowing 3 M KCl bridge. Compensation for series resistance (75–80%) and capacitance were performed electronically. Membrane currents were normalized for cell capacitance (pA/pF).

Current-voltage (I-V) relationships for I_to were determined by applying test pulses from −40 mV to +60 mV (400-ms duration) at a cycle length (CL) of 5 s. Typically, each test pulse was preceded by a prestep from a holding potential of −80 mV to −40 mV (duration 40 ms) to inactivate sodium current. Zero-sodium bathing solution was used in some experiments to block sodium current. For each test pulse the amplitude of I_to was measured as the difference between peak outward current and the current level at the end of the depolarizing clamp pulse. This clamp protocol was applied first in the control solution and then after 2 min in the acidic test solution. Each cell was exposed only once to an acidic test solution.

I-V relationships for I_Ca,L in myocytes were determined with the same protocol (CL = 5 s). At the end of the experiment the protocol was repeated in the presence of 200 μM CdCl₂ and 10 μM nifedipine to correct for background current.

I-V relationships for hKv4.3 and hKv1.4 expressed in HEK 293T cells were determined by holding the cell at a potential of −80 mV and applying test pulses from −40 mV to +60 mV (500-ms duration) at CL of 5 s and 30 s, respectively, and the clamp protocol was applied first in the control solution and then after 1 min in the acidic test solution.

The steady-state voltage dependence of I_to activation in myocytes was determined with results obtained from the I-V curve clamp protocol. Relative conductance (G/G_max) was calculated as G = I_to/(V_m − V_rev), where V_m is the clamp potential and V_rev is the estimated reversal potential (−76 mV was used as the estimated reversal potential, V_rev was calculated by Nernst equation and liquid junction potential). The relationship was fit to a Boltzmann function, and G_max was estimated by extrapolation of the curve to more positive potential. The resulting normalized G-V relationships were again fit with a Boltzmann function: G/G_max = 1/{1 + exp[(V_m − V_1/2)/k]}, where V_1/2 and k are the half-maximal activation potential and the slope (mV) of the curve, respectively.

The steady-state voltage dependence of I_to inactivation in myocytes was determined with a double-pulse protocol. Conditioning clamp pulses ranging from −80 mV to 0 mV (500-ms duration) were initiated from a holding potential of −80 mV at a CL of 5 s. At the end of each conditioning pulse V_m was stepped to −40 mV for 10 ms before application of the test pulse to +60 mV. I_Ca,L is negligible at +60 mV and thus is unlikely to contaminate measurements of I_to (41). Peak currents elicited by the test pulses were normalized as I_to/I_to,max and plotted as a function of conditioning V_m. The curves were fit with a Boltzmann function: I_to/I_to,max = 1/{1 + exp[(V_m − V_1/2)/k]}, where V_m is the conditioning voltage, and V_1/2 and k are the half-maximal inactivation potential and the slope (mV) of the curve, respectively.

The clamp protocol for quantifying the time course of I_to inactivation was the same as that used to generate I-V curves. With Clampfit software, the time course of inactivation of I_to was fit with a double-exponential function as previously described (22, 45) according to

$$I=A_{\text{f}}{e}^{-t/{\tau }_{\text{f}}}+A_{\text{s}}{e}^{-t/{\tau }_{\text{s}}}+C$$ (1)

where I is the current (pA/pF) at time t (ms), A_f (pA/pF) and A_s (pA/pF), respectively, are the amplitudes of fast and slow components, τ_f and τ_s, respectively, are the fast and slow inactivation time constants (ms), and C is the current remaining at the end of the clamp step (pA/pF).

The time course of recovery from I_to inactivation was determined with a double-pulse protocol at a CL of 10 s. A conditioning clamp pulse (P₁) to +60 mV (400-ms duration) was applied from a holding potential of −80 mV, followed at a variable interval ranging from 100 ms to 5 s by the test pulse (P₂) of 400-ms duration to +60 mV. All experiments were performed without Cd²⁺. Sodium-free bathing solution was used to block I_Na in the extracellular acidosis experiments. For the intracellular acidosis experiments, I_Na was inactivated by application of a prepulse from −80 mV to −40 mV (duration 40 ms) before clamping to +60 mV. The recovery from inactivation of I_to was fit with a single exponential as previously described (45) according to

$$I=A{e}^{-t/\tau }+C$$ (2)

where I is the current (pA/pF) at time t (ms), A (pA/pF) is the amplitude, and C is the current remaining at the end of the clamp step (pA/pF).

Action Potential Voltage Clamps

AP voltage-clamp experiments (AP clamp) were performed to assess the effects of intracellular acidosis on I_to under more physiological conditions than with rectangular clamp pulses. The bathing and pipette solutions used for these experiments were the same as those used to generate the I-V curve, except that all of the bathing solutions also contained 2 μM E-4031 to block I_Kr. AP templates (control and intracellular acidosis) were made from representative records obtained during current-clamp experiments. Each AP clamp was preceded by a prestep from −80 mV to −40 mV (40-ms duration) at a CL of 5 or 2 s. A train of 10 conditioning clamps was first applied in the control solution with the control AP template. They were then repeated after 2 min in the test solution with the intracellular acidosis AP template. The entire protocol was then repeated in the presence of 3 mM 4-aminopyridine to block I_to. The difference current was used to determine I_to flowing during the AP.

A very similar protocol was used to measure I_Ca,L in Epi and PM myocytes during AP clamps. AP templates (control and intracellular acidosis) were made from representative records obtained during current-clamp experiments. All measurements were performed in the control bathing solution. Ten conditioning AP clamp pulses were applied to the cells prior to the test clamp to ensure steady-state loading of SR Ca²⁺ when I_Ca,L and CaTs were recorded simultaneously (see Table 2) and when only CaTs were recorded (see Fig. 12). The control AP template served as the conditioning pulse for the control test clamp, and the intracellular acidosis AP template served as the conditioning pulse for the acidosis test clamp. The entire protocol was then repeated in the presence of 200 μM CdCl₂ and 10 μM nifedipine to correct for background current.

Measurement of Intracellular pH

pH_i was measured in single resting myocytes and HEK 293T cells with carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1), as previously described (2).

Measurement of Intracellular Calcium

CaTs were detected in single myocytes with a similar epifluorescence system using the fluorescent indicator fluo-4. Cells were incubated in the normal control solution containing 10 μM fluo-4 AM (Molecular Probes) and 0.3 mM probenicid at 25°C for 15 min. They were then continuously bathed in the same solution containing no indicator. Probenicid (0.3 mM) was included in the bathing solutions to help retard fluo-4 loss from the cells. Fluorescence emission (535 ± 11 nm, band-pass filter) was collected with a photomultiplier tube via the ×40 oil objective (numerical aperture 1.3) during continuous excitation at 485 nm with a 150-W xenon lamp. Fluorescence signals were background corrected and expressed as F/F₀ (the ratio of fluorescence during the CaT divided by diastolic fluorescence). CaT duration was measured as the time from the upstroke to 50% recovery (CaTD₅₀). CaTs were elicited with AP clamps (CL = 2 s) during superfusion with the normal bathing solution, using the appropriate AP templates for control and intracellular acidosis. A train of at least 10 conditioning clamps was applied to the cells to achieve steady-state Ca²⁺ loading of the SR. The normal pipette filling solution was used (no Cs⁺, no BAPTA) when only CaTs were measured. For experiments in which CaTs and I_Ca,L were simultaneously recorded, K⁺ in the bathing solution was replaced with Cs⁺ and the pipette filling solution was the normal solution used for I_Ca,L measurements except that it did not contain BAPTA. The gain of E-C coupling was calculated as the maximum rate of rise of the CaT divided by the simultaneously recorded peak I_Ca,L, as previously described (16).

Statistics

Summarized results are expressed as means ± SE. A paired Student's t-test was used to test significance between results obtained with each cell serving as its own control. An unpaired t-test was used to test significance between two different groups of cells. P < 0.05 was considered significant.

RESULTS

Changes in pH_i During Extra- and Intracellular Acidosis

Figure 1, A and B, illustrate the changes in pH_i in rabbit ventricular myocytes resulting from 2 min of extracellular and intracellular acidosis, respectively. HCO₃⁻-dependent transporter activity was minimized by superfusing with CO₂/HCO₃⁻-free solution, while NHE activity was blocked with 30 μM cariporide. As we have previously shown in rabbit ventricular myocytes (41), 2 min of low pH_o caused negligible changes in pH_i, reducing it by only ∼0.05 units (Fig. 1, A and D). In contrast, intracellular acidosis induced by superfusion with 80.0 mM acetate (pH_o 7.4) for 2 min markedly reduced pH_i by 0.47 units from a mean value of 7.22 ± 0.02 to 6.75 ± 0.04 (n = 17; Ref. 41). We have also found that pH_i is unaffected by attachment with a suction pipette filled with the normal solution (pH_pipette = 7.2; Ref. 41). The 2-min acidosis protocol was used for both types of acidosis in all subsequent myocyte experiments.

Fig. 1.

Changes in intracellular pH (pH_i) and intracellular H⁺ concentration ([H⁺]_i) induced by extra- and intracellular acidosis and by pipette acid loading. A: a reduction in extracellular pH (pH_o) from 7.4 to 6.5 for 2 min elicited only a small drop in pH_i. B: in contrast, exposure to 80.0 mM acetate (pH_o 7.4) induced a rapid sustained fall in pH_i. C: example record showing the marked difference between intracellular acidosis (pH_o 7.4) induced with 80 mM acetate and with pipette dialysis [pipette pH (pH_p) = 6.6]. D: summarized results for extracellular acidosis (n = 7), intracellular acidosis (n = 17), and pipette acid loading (n = 4). We previously reported the summarized results shown in D for extra- and intracellular acidosis (41). The example records in A and B have not been previously published. All results shown here were obtained from a mixture of cells from all regions of the left ventricle. **P < 0.01 paired, control vs. acidosis. In separate experiments, we found no significant difference between the drop in pH_i induced by 80.0 mM acetate (pH_o 7.4) in epicardial (Epi, n = 4) and papillary muscle (PM, n = 7) myocytes (data not shown).

Several earlier studies used pipette acid loading to induce intracellular acidosis in cardiac myocytes, on the assumption that pH_i equilibrates with the pH of the pipette filling solution (25, 29, 58, 59). However, these studies did not include measurements of pH_i. Figure 1C shows that 10 min of intracellular dialysis with a pipette pH of 6.6 induced a fall in the measured pH_i of only 0.17 units, from 7.20 ± 0.04 to 7.03 ± 0.04 (n = 4). When expressed as change in intracellular H⁺ concentration ([H⁺]_i), this represents an ∼1.5× increase compared with 3.1× increase induced by the 80.0 mM acetate pulse. Thus, assuming that pH_i = pH_pipette will seriously overestimate the degree of intracellular acidosis and emphasizes the importance of measuring pH_i as we have done in the present study.

Effect of Acidosis on Action Potential Repolarization in Epicardial Myocytes

Because we used myocytes from all regions of the left ventricle in our previous study of pH effects on AP repolarization, there were large cell-to-cell differences in phase 1 and the notch (41). In this series of experiments we focused on the AP response of Epi myocytes to extracellular and intracellular acidosis. As shown in Fig. 2, epicardial APs display a prominent phase 1 repolarization and notch, mediated primarily by the high density of I_to displayed in this cell type (17).

Fig. 2.

Effect of extra- and intracellular acidosis on epicardial action potentials (APs). A: extracellular acidosis (pH_o 6.5) shortened AP duration (APD). Inset: expanded view of phase 1 repolarization. B: intracellular acidosis (80.0 mM acetate) prolonged APD, markedly slowed phase 1 repolarization, and nearly eliminated the notch. Inset: expanded view of phase 1 repolarization. C: summarized changes in APD at 90% repolarization (APD₉₀) in low pH_o (n = 5) and low pH_i (n = 11). D: changes (Δ) in APD₉₀ shown in C expressed as % relative to control. The normal pipette filling solution was used in these experiments (no BAPTA). Pacing cycle length (CL) for all experiments was 2 s. *P < 0.05, **P < 0.01, paired, control vs. acidosis.

In response to 2 min of extracellular acidosis, the plateau was depressed and APD was shortened but the notch was largely unaffected (Fig. 2, A, C, and D). The resting membrane potential (CL = 2 s) was slightly depolarized by extracellular acidosis (control = −87.1 ± 2.5 mV, acidosis = −83.8 ± 2.7 mV; n = 5, P < 0.05, paired). The effects of extracellular acidosis on cardiac AP configuration are complex, with reports of inhibition of the rapid delayed-rectifier current I_Kr (8), I_Ca,L (8, 41), and both peak and persistent sodium current (30). In addition, we have recently shown that the changes in rabbit ventricular repolarization induced by low pH_o are largely mediated by a reduction in I_Ca,L and are unlikely to involve I_to (41).

In contrast, intracellular acidosis markedly slowed phase 1 repolarization, reduced the notch, elevated the plateau, and prolonged APD (Fig. 2, B–D; pacing CL = 2 s). The percent change in APD₉₀ induced by low pH_i at CL = 2 s (+88.5 ± 9.0%, n = 11) was not significantly different from that at CL = 5 s (+86.2 ± 21.0%, n = 4). The resting membrane potential (CL = 2 s) was slightly depolarized by low pH_i (control = −81.0 ± 1.0 mV, acidosis = −79.0 ± 1.5 mV; n = 11 P < 0.05, paired), but the overshoot did not change (control = 45.7 ± 1.2 mV, acidosis 45.3 ± 1.3 mV; n = 11).

We also preformed AP measurements (CL = 2s) with 10 mM BAPTA in the suction pipette to minimize Ca²⁺-activated Cl⁻ current (I_Cl,Ca). This concentration of BAPTA blocks CaTs and the rise in diastolic calcium induced by lowering pH_i (41). With BAPTA dialysis the changes in AP repolarization during both types of acidosis were qualitatively the same as those without BAPTA and included AP shortening in low pH_o and AP lengthening in low pH_i, suggesting that changes in I_Cl,Ca did not play a major role [intracellular acidosis: control APD₉₀ = 414 ± 25 ms, acidosis APD₉₀ = 922 ± 36 ms (n = 2, P < 0.02, paired); extracellular acidosis: control APD₉₀ = 585 ± 50 ms, acidosis APD₉₀ = 519 ± 53 ms (n = 3, P < 0.05, paired)].

Numerous studies have demonstrated that I_to inhibition slows phase 1 repolarization, elevates the plateau, and reduces the notch (4, 17, 18, 39). Thus the results in Fig. 2 strongly suggest that the I_to activated during APs is inhibited by intracellular acidosis but largely unaffected by extracellular acidosis.

Effect of Acidosis and Cadmium on Steady-State Voltage Dependence of I_to Inactivation

In this series of experiments we examined the separate actions of extra- and intracellular acidosis on the voltage dependence of I_to inactivation in Epi myocytes (Fig. 3). We also assessed the effect of Cd²⁺ on inactivation since it was used in other protocols to block I_Ca,L. After each 500-ms-duration conditioning step, V_m was clamped to +60 mV, which is near the peak of the typical rabbit ventricular AP (Fig. 1) and activates a large I_to with minimal contamination by I_Ca,L.

Fig. 3.

Effect of acidosis and Cd²⁺ (200 μM) on the steady-state voltage dependence of transient outward current (I_to) inactivation in Epi myocytes. Ai: extracellular acidosis (pH_o 6.5) without Cd²⁺ induced a right shift in inactivation (n = 4). Cd²⁺ right-shifted inactivation and reduced the shift induced by extracellular acidosis (n = 4). Aii: control and acidosis signals from the same cell (no Cd²⁺). Red signals clamped from −40 mV. Bi: intracellular acidosis (80.0 mM acetate) with (n = 5) or without (n = 4) Cd²⁺ had no significant effect on the voltage dependence of inactivation. Bii: control and acidosis signals from the same cell (no Cd²⁺). Red signals clamped from −40 mV. Smooth lines are best fits with Boltzmann functions (materials and methods). Inset: clamp protocol. The results in this figure are summarized in Table 1. Pacing CL = 5 s.

Previous work with rat ventricular myocytes, measured at room temperature, reported that both extracellular acidosis (50) and simultaneous reductions in pH_o and pH_i (24) induced right shifts in the voltage dependence of I_to inactivation. Our results, measured at 37°C without Cd²⁺ or dihydropyridine Ca²⁺ channel blockers, confirm that extracellular acidosis (pH_o 6.5) has the same effect in rabbit epicardial ventricular myocytes, shifting the inactivation V_1/2 by ∼10 mV (Fig. 3A; Table 1, no Cd²⁺). The same result was also obtained in rabbit cells bathed in sodium-free solution containing no Cd²⁺ (Table 1). In accord with earlier work in rat and rabbit myocytes (1, 50, 55), we also found that Cd²⁺ (200 μM) right-shifted the voltage dependence of I_to inactivation (Fig. 3A; Table 1, compare control no Cd²⁺ with control 200 μM Cd²⁺). In addition, the H⁺-induced right shift was less in the presence of Cd²⁺ (Fig. 3A), as also observed in rat ventricular myocytes (50).

Table 1.

Effect of acidosis and Cd²⁺ on I_to steady-state inactivation parameters

	200 mM Cd2+		No Cd2+
	Extracellular acidosis (n = 4)	Intracellular acidosis (n = 5)	Extracellular acidosis (n = 4)	Intracellular acidosis (n = 4)	No Na+, No Cd2+ Extracellular Acidosis (n = 5)
Control V1/2, mV	−34.6 ± 1.8	−34.3 ± 1.8	−44.4 ± 1.6	−38.6 ± 2.1	−43.5 ± 0.4
Acidosis V1/2, mV	−28.7 ± 1.6**	−34.4 ± 2.4	−34.5 ± 2.5**	−38.2 ± 1.6	−29.2 ± 1.4**
Control slope k	5.4 ± 0.3	5.7 ± 0.3	5.6 ± 0.7	4.4 ± 0.2	10.3 ± 0.5
Acidosis slope k	5.6 ± 0.2	7.1 ± 0.4	5.1 ± 0.4	5.3 ± 0.5	8.8 ± 0.4*

Values are mean ± SE half-maximal inactivation potential (V_1/2) and k determined from best fits of Boltzmann functions. I_to, transient outward current.

^*P < 0.05,

^**P < 0.01 vs. control, paired.

In striking contrast to low pH_o, intracellular acidosis had no significant effect on the voltage dependence of I_to inactivation with or without Cd²⁺ in the bathing solution (Fig. 3B; Table 1). This indicates that Cd²⁺ can be used to block I_Ca,L in order to study the effects of changes in pH_i on the steady-state gating properties of I_to. Importantly, it also suggests that low pH_i does not affect steady-state inactivation of I_to. This lack of effect has been reported for rat ventricular myocytes measured at room temperature (58, 59). However, pipette acid loading was used in that study to induce intracellular acidosis without measuring pH_i, which our results demonstrate (Fig. 1, C and D) is not a reliable method for inducing quantitative changes in pH_i.

Influence of Voltage Clamp Prestep and Cd²⁺ on Response of I_to Activation to Acidosis

It is necessary to block the contaminating effects of I_Ca,L and I_Na when assessing the response of I_to activation to acidosis. I_Ca,L can be blocked with Cd²⁺, and I_Na is readily inactivated by applying a 40-ms prestep from −80 mV to −40 mV. Here we assess the use of these two approaches for studying the effects of acidosis on I_to activation.

Low pH_o.

The action of both low pH_o and external Cd²⁺ to right-shift I_to inactivation makes it difficult to accurately assess the voltage dependence of I_to activation during extracellular acidosis with Cd²⁺ present. To avoid using Cd²⁺, we examined the effect of low pH_o on I_to activation in Epi myocytes clamped to +60 mV, which, as noted above, is near the peak of the AP and is a voltage at which I_Ca,L is negligible (41). To assess the effects of a prestep on I_to activation, we used sodium-free solutions to block I_Na. Figure 4A shows that extracellular acidosis had no effect on I_to in the absence of external sodium, external Cd²⁺, and a prestep. In contrast, when the prestep was included I_to increased during low pH_o (Fig. 4B), presumably because of increased channel availability at −40 mV (Fig. 3A). The results are summarized in Fig. 4C. Thus inclusion of even a brief prestep will introduce errors in the measurement of I_to activation during extracellular acidosis.

Fig. 4.

Effect of a voltage clamp prestep on the response of I_to activation to extracellular acidosis (pH_o 6.5) in Epi myocytes. A: example experiment showing that, without a prestep to −40 mV, low pH_o has no effect on I_to activated by a voltage clamp step from −80 mV to +60 mV. B: example experiment (same cell as A) showing that low pH_o increased I_to when a 40-ms-duration prestep from −80 mV to −40 mV was included. C: summary of the results, expressed as % change in I_to relative to control with and without the prestep (n = 4). All experiments in this figure were performed in sodium- and Cd²⁺-free solution. Pacing CL = 5 s. **P < 0.01 paired, with vs. without prestep.

Most importantly, these results suggest that extracellular acidosis does not significantly affect I_to in rabbit epicardial ventricular APs when they are initiated from normal resting potentials and have normal overshoots. In most subsequent experiments we focused on intracellular acidosis.

Low pH_i.

In the next series of experiments we examined the effect of Cd²⁺ (200 μM) and the 40-ms prestep to −40 mV on I_to activation in Epi myocytes during intracellular acidosis (Fig. 5). As in Fig. 4, the cells were clamped from a holding potential of −80 mV to +60 mV to minimize I_Ca,L. Intracellular acidosis markedly reduced I_to in each of the four conditions: Na⁺ plus Cd²⁺ (Fig. 5Aa); Na⁺, no Cd²⁺ (Fig. 5Ab); no Na⁺, no Cd²⁺ (Fig. 5Ac); and no Na⁺, no Cd²⁺, and no prestep (Fig. 5Ad). The results are summarized in Fig. 5B and show that neither the presence of Cd²⁺ nor the prestep significantly affected the action of low pH_i to reduce I_to.

Fig. 5.

Effect of a voltage clamp prestep and Cd²⁺ (200 μM) on the response of I_to activation to intracellular acidosis (80.0 mM acetate) in Epi myocytes. A: example experiment showing that, in the presence of the 40-ms prestep and normal external sodium, low pH_i reduced I_to both with (a) and without (b) the presence of Cd²⁺ and example signals from a cell showing that, in sodium- and Cd²⁺-free solution, low pH_i reduced I_to both with (c) and without (d) the 40-ms prestep. B: summary of results, expressed as % change in I_to relative to control. There were no significant differences among these groups (ANOVA); n = 9 with prestep, normal sodium, and Cd²⁺; n = 4 with prestep, normal sodium, no Cd²⁺; n = 4 with prestep, no sodium, no Cd²⁺; n = 4 with no prestep, no sodium, no Cd²⁺. Pacing CL = 5 s.

Effect of Intracellular Acidosis on I_to Activation and Kinetics of Inactivation

On the basis of the results in Fig. 5, we further examined the voltage dependence of I_to activation in Epi myocytes during intracellular acidosis, using a prestep with both Cd²⁺ (200 μM) and normal sodium in the bathing solutions (Fig. 6). As shown in the example signals in Fig. 6A and summarized in Fig. 6B, intracellular acidosis (pH_o 7.4, pH_i 6.75) reduced I_to by ∼50% at nearly all voltages between −40 mV and +60 mV. The percent reduction in I_to at +40 mV with CL = 5 s (−56.2 ± 3.1%) was not significantly different from that at CL = 1 s (−61.1 ± 3.8%; n = 5, paired).

Fig. 6.

Effect of intracellular acidosis on I_to activation in Epi myocytes. A: example records from same cell showing the inhibitory action of intracellular acidosis (80.0 mM acetate) on I_to. Inset: voltage-clamp protocol. B: summary of results illustrating depressed current-voltage (I-V) curve at nearly all voltages (n = 9; **P < 0.01 paired, control vs. acidosis). C: intracellular acidosis (80.0 mM acetate) had negligible effects on steady-state activation and inactivation of I_to. Inactivation results same as shown in Fig. 3B (with Cd²⁺). Smooth lines are best fits with Boltzmann functions (see materials and methods). D: quantitative relationship between I_to and pH_i in Epi myocytes. Data fit with the equation I_to = a + b/{1+[10^(pK−pH)]ⁿ} (40). The respective values for a, b, pK, and n were 2.81, 8.523, 6.92, and 3.12. Inset: clamp protocol. pH_i measurements were done in separate cells, with N values ranging from 5 to 13 and are from Saegusa et al. (41). The magnitude of intracellular acidosis (pH_o 7.4) was varied by applying different concentrations of sodium acetate for 2 min while keeping extracellular Ca²⁺ activity constant. The lowest value of pH_i was induced with 80.0 mM acetate. E: effect of low pH_i (80.0 mM acetate) on the voltage dependence of I_to inactivation kinetics. Cells were voltage clamped according to the protocol shown in A. Low pH_i decreased fast time constant of I_to inactivation (τ_f) at all voltages (n = 5; **P < 0.01 paired, *P < 0.05, paired, control vs. acidosis). τ_s, slow time constant of I_to inactivation. For all results shown in this figure pacing CL = 5 s; Cd²⁺ (200 μM) and normal sodium were present in all bathing solutions.

Figure 6C summarizes the effects of intracellular acidosis on both steady-state activation and inactivation. Low pH_i did not significantly change the V_1/2 of activation (11.3 ± 1.5 mV for control vs. 11.4 ± 1.9 mV for acidosis), while the slope was significantly decreased from −19.7 ± 0.8 mV in control to −15.3 ± 0.8 mV (n = 9, P < 0.01 paired). Steady-state inactivation was also unaffected by intracellular acidosis (with Cd²⁺, same results shown Fig. 3B).

The quantitative relationship between pH_i (pH_o 7.4) and epicardial I_to at +50 mV is summarized in Fig. 6D and shows that I_to was inhibited by low pH_i with an apparent pK of 6.92.

Figure 6E summarizes the voltage dependence of the fast (τ_f) and slow (τ_s) time constants of I_to inactivation from +40 mV to +60 mV (CL = 5 s). Low pH_i had no significant effect on τ_s but speeded τ_f at all voltages. Similarly, when the CL was maintained at 2 s, low pH_i had no significant effect on τ_s but reduced τ_f from 10.4 ± 0.5 ms to 6.3 ± 0.6 ms at +50 mV (n = 4, P < 0.01, paired).

Effect of Acidosis on Recovery from Inactivation

The time course of recovery from inactivation in Epi myocytes was studied with a standard two-pulse protocol (Fig. 7). The low-pH_o experiments were performed in sodium-free solution without a prestep. The recovery from inactivation of I_to in myocytes from control and both two types of acidosis was best fit by a single-exponential function. The recovery time constant was unaffected by low pH_o but slowed by low pH_i.

Fig. 7.

Effect of acidosis on the time course of I_to recovery from inactivation. A: extracellular acidosis (pH_o 6.5) had no significant effect on the time constant of recovery from inactivation (τ_cont = 986 ms, τ_acid = 1,009 ms; n = 4). a and b: Example signals from the same cell. c: Summarized results. B: in contrast, low pH_i (80.0 mM acetate) slowed recovery from inactivation (τ_cont = 1,421 ms, τ_acid = 2,997 ms; n = 4). a and b: Example signals from the same cell. c: Summarized results. Clamp CL = 10 s. Insets: averaged actual values of I_to (pA/pF) in cells clamped with test pulses (P₂) to +60 mV after conditioning clamp pulse (P₁) to +60 mV.

Effect of Intracellular Acidosis on I_to During AP Clamps

To determine the effects of low pH_i on Epi I_to under more physiological conditions than with rectangular clamp steps, we also conducted AP clamp experiments (Fig. 8). Clamps were applied to Epi myocytes at CLs of 2 and 5 s since I_to and phase 1 repolarization in rabbit ventricular myocytes are affected by pacing rate (17, 20). The templates used for the control and acidosis AP clamps were representative of epicardial APs recorded at both rates. Figure 8A illustrates the inhibitory effect of low pH_i on I_to elicited by AP clamps (CL = 5 s). The peak amplitude of I_to was significantly reduced at both pacing rates (Fig. 8B), and there was no significant difference in the percent change relative to control between the two rates (unpaired). These results demonstrate the striking action of internal protons to inhibit I_to during APs.

Fig. 8.

Effect of intracellular acidosis on Epi I_to during AP clamps. A: example AP clamp experiment showing the action of intracellular acidosis (80.0 mM acetate) to reduce I_to. CL = 5 s. B: summary of results for peak I_to during AP clamps (CL = 5 s, n = 8; CL = 2 s, n = 7). **P < 0.01, paired, compared with control.

Effect of Acidosis on Human Kv4.3 and Kv1.4 Expressed in HEK 293T Cells

On the basis of the voltage-dependent kinetics of recovery from inactivation and the time course of inactivation, transient outward current can be classified as fast (I_tof) and slow (I_tos). I_tof shows rapid recovery (i.e., recovery time constants on the order of 10 to hundreds of milliseconds), and I_tos shows slow recovery (i.e., on a timescale of seconds) (36). Evidence indicates that the pore-forming domain of the channel underlying I_tof is formed by a combination of Kv4.2 and/or Kv4.3 subunits, while Kv1.4 is the molecular component underlying I_tos (32, 34, 36). In addition, a variety of regulatory subunits have major effects on the properties of Kv1.4, Kv4.2, and Kv4.3, including channel kinetics and expression (32, 36). Although the exact molecular basis of I_to in rabbit ventricle is unresolved, expression of mRNA and/or protein of Kv4.2, Kv4.3, and Kv1.4 has been reported (34, 39, 54).

To help identify which channel mediates the response of rabbit I_to to low pH_i, we examined the effects of intracellular acidosis on hKv4.3 and hKv1.4 channels expressed in HEK 293T cells (Fig. 9). The bathing solutions contained normal sodium and no Cd²⁺, and no prestep was required to block sodium current. As shown in Fig. 9A, exposure to 40 mM acetate for 1 min (pH_o 7.4, 30 μM cariporide) rapidly reduced pH_i from a control value of 7.26 ± 0.01 to 6.70 ± 0.04 (n = 4), which is comparable to that in ventricular myocytes bathed in 80 mM acetate (Fig. 1D). This difference in response to acetate (40 mM vs. 80 mM) most likely reflects a lower intrinsic buffering power in HEK cells. Low pH_i decreased hKv4.3 current at nearly all voltages but had no significant effect on the I-V relationship of hKv1.4 (Fig. 9B). In contrast to the effects of low pH_i, extracellular acidosis had no significant effect on the I-V curve of hKv4.3 over the voltage range of −40 mV to +60 mV (n = 3, data not shown).

Fig. 9.

Effects of intracellular acidosis on human (h)Kv4.3 and hKv1.4 channels expressed in HEK 293T cells. A: example of pH_i measurement in a nontransfected HEK 293T cell showing the effects of a 1-min exposure to 40.0 mM acetate (pH_o 7.4). B, a: low pH_i depressed the I-V curve for hKv4.3 at nearly all voltages (n = 4). Inset: example experiment showing the inhibitory effect of low pH_i on hKv4.3 current elicited at +60 mV. b: In contrast, the I-V curve for hKv1.4 current was unaffected by low pH_i (n = 4). Inset: example experiment showing the lack of effect of low pH_i on peak hKv1.4 current elicited at +60 mV. *P < 0.05, paired, control vs. acidosis.

The kinetics of inactivation of both hKv4.3 and hKv1.4 were accelerated by intracellular acidosis. Thus for Kv4.3 inactivation, two of the four cells were best fit by a single exponential and yielded time constants of inactivation at +60 mV of τ_control = 163 ± 3 ms and τ_acidosis = 110 ± 10 ms. The same effect also occurred in the two cells best fit by a double exponential (τ_fast,control = 18 ± 4 ms, τ_{fast,acidosis} = 11 ± 3 ms; τ_slow,control = 134 ± 12 ms, τ_{slow,acidosis} = 124 ± 10 ms; +60 mV). The time course of hKv1.4 inactivation at +60 mV was best fit by a single exponential and yielded a τ_control = 108 ± 25 ms and τ_acidosis = 76 ± 17 ms (n = 4, P < 0.05, paired).

Taken together, the results in Fig. 9 suggest that the inhibition of I_to by intracellular acidosis in rabbit ventricular myocytes is mediated, in part, by blockade of Kv4.3 current.

Effect of Intracellular Acidosis on AP and I_to in Papillary Muscle Myocytes

Rabbit ventricular myocytes display marked regional differences in I_to density, with Epi cells having the highest and PM cells the lowest (17). This current gradient has large effects on AP repolarization in the intact heart. Given this spatial heterogeneity, it was of interest to also study the response of PM myocytes to intracellular acidosis.

Intracellular acidosis had little effect on phase 1 repolarization in PM cells, and the prolongation of APD was much less than in Epi myocytes (Fig. 10, A and B). Figure 10C demonstrates that under control conditions I_to current density in PM myocytes was approximately half that of Epi cells, in accord with previous work (17). During intracellular acidosis I_to was reduced by ∼50% at all voltages in PM cells (Fig. 10, C and D). These results suggest that regional differences in I_to current density make a significant contribution to the differential response of AP repolarization to low pH_i in Epi and PM myocytes.

Fig. 10.

Effects of intracellular acidosis on AP and I_to in PM myocytes. A: example experiment showing the effect of intracellular acidosis (80.0 mM acetate) to prolong APD with little effect on phase 1 repolarization. CL = 2 s. Inset: expanded view of phase 1 repolarization. B, a: summarized APD₉₀ results for low pH_i in PM myocytes at CL = 2 s (n = 8; **P < 0.01, paired). b: % change in APD₉₀ relative to control after 2 min of low pH_i in Epi (n = 11; same results as Fig. 2D) and PM (n = 8; **P < 0.01, unpaired) myocytes. C: low pH_i in PM myocytes (n = 8) depressed I_to at nearly all voltages. CL = 5 s. **P < 0.01, paired. Control I-V curve from Epi myocytes shows same data as Fig. 6B. D: summary of results for I_to in Epi (n = 9) and PM (n = 8) myocytes at +50 mV and % change relative to control. CL = 5 s. ^##P < 0.01, unpaired, comparison of control I_to amplitude in Epi and PM myocytes; **P < 0.01, paired, control vs. acidosis.

Modulation of I_Ca,L by pH_i-Induced Changes in AP Waveform

The effects of intracellular acidosis on AP repolarization in Epi myocytes are much more prominent than those in PM cells (compare Fig. 2B to Fig. 10A). Epi cells show a marked slowing of phase 1, loss of the notch, and much greater AP prolongation (Fig. 10Bb). Since phase 1, the notch, and APD have been shown to modulate I_Ca,L, this could have significant effects on calcium handling (42, 43). In this series of experiments we sought to determine how I_Ca,L is affected by pH_i-induced changes in AP repolarization in Epi and PM myocytes, using AP voltage clamps. Only male rabbits were used for these experiments since transmural differences in peak I_Ca,L density have been reported for female rabbits (37). To confirm the absence of a transmural gradient we measured I_Ca,L density in both cell types under control conditions (no acidosis) with a conventional rectangular voltage-clamp protocol. As shown in Fig. 11A, the I-V curves for I_Ca,L were virtually identical in the two cell types.

Fig. 11.

Response of L-type calcium current (I_Ca,L) in Epi and PM myocytes to changes in AP repolarization induced by intracellular acidosis (80.0 mM acetate). Control bathing solution was used for all experiments shown in this figure. A: I_Ca,L in control bathing solution was virtually the same in Epi (n = 11) and PM (n = 9) cells. B, a: example of an AP clamp experiment (Epi myocyte) showing the action of the intracellular acidosis template to decrease peak I_Ca,L and prolong its duration. b: Example of an AP clamp experiment (PM myocyte) showing the action of the intracellular acidosis template to prolong I_Ca,L duration with little effect on peak current. C: summary of results for peak I_Ca,L during AP clamps (Epi n = 11, PM n = 9; **P < 0.01, paired, control vs. acidosis template). D: summary of results for net Ca²⁺ influx via I_Ca,L during AP clamps. Same cells as in C. E: results in C and D expressed as % change. ^##P < 0.01, unpaired, Epi vs. PM. CL = 2 s was used for all experiments shown in this figure.

For AP voltage-clamp experiments both the control and acidosis templates were applied in the control bathing solution (K⁺ replaced with Cs⁺) with the normal pipette filling solution for I_Ca,L measurements (Cs⁺, BAPTA). This approach allowed us to determine how pH_i-induced changes in AP repolarization per se affect I_Ca,L without the complicating effects of intracellular acidosis.

As illustrated in Fig. 11, B–E, application of the acidosis template decreased the initial peak value of I_Ca,L and prolonged its duration, with the greatest changes occurring in Epi cells. Despite the fall in peak I_Ca,L (Fig. 11C), net Ca²⁺ influx via I_Ca,L increased in both cell types, with the greatest increase occurring in Epi cells (Fig. 11D). The changes in peak I_Ca,L and net Ca²⁺ influx, expressed as percentage, are summarized in Fig. 11E.

These results demonstrate that the action of low pH_i to slow phase 1 repolarization and prolong APD significantly alters I_Ca,L and net Ca²⁺ influx via I_Ca,L, with the greatest changes occurring in Epi myocytes. Thus internal H⁺ ions can have significant indirect effects on voltage-dependent I_Ca,L mediated, in part, by pH_i-induced changes in I_to that alter the time course of repolarization.

Modulation of CaTs by pH_i-Induced Changes in AP Waveform

To determine whether CaTs elicited by APs are affected by pH_i-induced slowing of phase 1 repolarization and APD prolongation, we performed the AP clamp experiments shown in Fig. 12. All studies were performed in control bathing solution with the same AP templates as those shown in Fig. 11 and the normal pipette filling solution (no Cs⁺, no BAPTA). The Epi templates were applied to Epi myocytes, and the PM templates were applied to PM myocytes. Small but significant increases in the amplitude and rate of rise of the CaT occurred in Epi but not PM myocytes (Fig. 12, Ba and Bb). The duration of the CaT (CaTD₅₀) was increased by the intracellular acidosis template in both cell types (Fig. 12, A and Bc).

We also performed separate AP clamp experiments in which I_Ca,L and CaT were simultaneously recorded in Epi and PM myocytes. The AP templates were the same as those shown in Fig. 12A and were applied to their respective cell types. Potassium in the bathing solution was replaced with Cs⁺, and the pipette filling solution was the normal solution used for I_Ca,L measurements, except that it contained no BAPTA. The results are summarized in Table 2 and show that the intracellular acidosis template decreased peak I_Ca,L by −31.0 ± 3.9% and −7.8 ± 1.7% in Epi (n = 5) and PM (n = 5) myocytes, respectively. These results are very similar to those obtained with AP clamps during BAPTA dialysis (Fig. 11C). In addition, the acidosis template in Epi but not PM cells increased both the amplitude and rate of rise of the CaT. This resulted in a significant increase in E-C coupling gain in Epi but not PM myocytes, calculated as maximum rate of rise of the CaT divided by peak I_Ca,L.

Collectively, these results demonstrate that the striking changes in AP repolarization induced by intracellular acidosis have a significant modulating influence on ventricular calcium handling that is especially evident in cells with a prominent I_to.

DISCUSSION

Changes in myocardial pH have multiple effects on electrical activity and Ca²⁺ signaling (14, 33). This sensitivity accounts in part for the arrhythmias and depression of ventricular function that occur during myocardial ischemia, a condition associated with a fall in both pH_i and pH_o (3, 19). Here we examined the response of rabbit ventricular I_to to acute extra- and intracellular acidosis, especially intracellular, and determined the resulting effects on AP repolarization. We also studied the indirect action of these repolarization changes on I_Ca,L and CaTs. In contrast to previous reports of pH effects on ventricular I_to (24, 50, 58, 59), our experiments were performed at physiological temperature with a myocyte preparation with a more humanlike AP than that of rat.

We demonstrate that I_to is rapidly and markedly reduced by intracellular acidosis (Figs. 6, 8, and 10). This occurred in cells with both high (Epi) and low (PM) I_to densities. In both cell types low pH_i reduced peak I_to over a large voltage range (−20 mV to +60 mV), resulting in an ∼50% fall in current density when pH_i is reduced from 7.22 and 6.75. The apparent pK of this relationship in Epi cells was 6.92, demonstrating the high H⁺ sensitivity of I_to over the physiological range of pH_i values.

Interestingly, this reduction in peak current was not accompanied by shifts in either steady-state inactivation or activation curves. This contrasts with the action of external H⁺ to induce right shifts (toward less negative potentials) in these parameters as reported here for rabbit ventricular myocytes (Fig. 3A) and previously for rat and human ventricular myocytes (50). These shifts presumably result from H⁺ screening of and/or binding to anionic sites such as carboxylic or amine/imidazole residues on the external sarcolemma and/or the channel itself. We have recently shown that both extra- and intracellular acidosis elicit large right and left shifts, respectively, in steady-state activation and inactivation of I_Ca,L in rabbit ventricular myocytes (41). The absence of shifts in I_to gating parameters during intracellular acidosis cannot be explained by the present study, but perhaps it reflects a low density of intracellular anionic sites on the channel itself.

Previous studies of rat ventricular myocytes also reported that intracellular acidosis reduced peak I_to without altering steady-state activation and inactivation (58, 59). However, those experiments were performed with pipette acid loading, without measuring pH_i, and in some experiments without the same cell serving as its own control. In addition, the origin of the cells was not specified, e.g., epicardial or endocardial. All of these issues were avoided in the present work.

The absence of voltage shifts in I_to gating, accompanied by a large reduction in peak current during low pH_i, strongly suggests a direct action of internal protons to reduce channel permeability, perhaps by titrating binding sites within the channel pore. This effect has a rapid onset and thus is unlikely to reflect a decrease in channel density.

We found no significant effect of extracellular acidosis on recovery from inactivation (Fig. 7A). In contrast, we did observe a small slowing of I_to recovery from inactivation during intracellular acidosis (Fig. 7B). However, this did not significantly affect the action of intracellular acidosis to reduce peak I_to during AP or rectangular voltage clamps elicited at CLs of 5, 2, or 1 s. Thus acidosis-induced slowing of recovery from inactivation does not appear to contribute significantly to the inhibitory effects of low pH_i on I_to.

Response of Kv4.3 and 4.1 Currents to Intracellular Acidosis

Previous studies using heterologous expression systems reported that extracellular acidosis inhibited both human Kv4.3 current (49) and Kv1.4 from rat and ferret (10). In contrast, human Kv1.4 current is reported to be unaffected by extracellular acidosis but inhibited by intracellular acidosis (35). Kv1.4 and Kv4.3 are both expressed in rabbit ventricle (34, 39, 54). To help identify the molecular basis for the inhibitory action of intracellular acidosis on rabbit I_to, we examined the effects of low pH_i on human Kv4.3 and Kv1.4 channels expressed in HEK 293T cells (Fig. 9). Kv1.4 current was unresponsive to a fall in pH_i from 7.3 to 6.7 (pH_o 7.4), while Kv4.3 current magnitude was significantly reduced over the voltage range of −20 to +60 mV (Fig. 9), suggesting that Kv4.3 mediates, in part, the sensitivity of rabbit ventricular I_to to pH_i. However, we cannot rule out the possibility that differences in regulatory subunits and temperature (37°C vs. 23°C) between the native channels and those expressed in HEK cells may also affect the pH_i sensitivity of these currents.

Relationship Between Action Potential Repolarization and I_to Inhibition by low pH

Consistent with the higher I_to density in Epi myocytes, the repolarization effects of intracellular acidosis to reduce I_to were most evident in this cell type, with marked slowing of phase 1, attenuation of the notch, and elevation of the plateau (compare Fig. 2B and Fig. 10B). The role of I_to inhibition in overall prolongation of APD in both cell types is less clear, but it seems likely that it contributed in part. Regardless of the detailed mechanism for the increased APD during low pH_i, the magnitude of AP prolongation in Epi cells was approximately twice that in PM cells.

Although extracellular acidosis induced a right shift in steady-state inactivation (Fig. 3A), it did not affect the magnitude of I_to at +60 mV, a voltage near the peak of the AP, when activated from V_m near the normal diastolic value (Fig. 4A). In addition, both extra- and intracellular acidosis had only small effects on diastolic V_m (Fig. 2). Thus it seems unlikely that I_to is involved in the pH_o-induced shortening of APD we observed in this preparation, a conclusion supported by our previous computer simulations (41).

Modulation of Calcium Handling by Intracellular Acidosis During Action Potentials

Our AP clamp experiments, performed with normal pH_o and pH_i, revealed that the changes in AP configuration induced by intracellular acidosis have significant effects on peak I_Ca,L, net Ca²⁺ influx via I_Ca,L, and the CaT (Fig. 11, Fig. 12, Table 2). These effects appear to be mediated, in part, by pH_i-induced changes in I_to and are more prominent in Epi than PM myocytes. Several earlier studies reported that changes in phase 1 repolarization significantly affect I_Ca,L and CaTs during APs (6, 11, 43, 44). However, to our knowledge, this is the first demonstration that changes in ventricular repolarization induced by low pH_i have significant indirect effects on calcium handling.

Our finding of a reduction in peak I_Ca,L, resulting from I_to inhibition (Fig. 11), is consistent with canine myocyte simulations (21) and rat myocyte experiments (11, 43, 44) and is likely mediated by the reduced voltage driving force acting on Ca²⁺ current. Epi myocytes displayed the greatest change in peak I_Ca,L, reflecting their higher I_to density and thus more pronounced slowing of phase 1. Despite the drop in peak I_Ca,L, net Ca²⁺ influx via I_Ca,L increased in both Epi and PM cells, presumably because of the prolonged APs. The largest increase in Ca²⁺ influx occurred in Epi cells, reflecting the greater AP lengthening (Fig. 11D).

The prolongation of the AP also caused CaT duration to increase in both cell types (Fig. 12Bc). However, the amplitude and rate of rise of the CaT as well as E-C coupling gain increased significantly only in Epi myocytes (Fig. 12, Ba and Bb, Table 2). These increases occurred despite a drop in the peak amplitude of Epi I_Ca,L. Under normal conditions, for a given magnitude of I_Ca,L, the amplitude of the CaT is steeply dependent on SR calcium load (46). It seems likely that the much larger increase in net Ca²⁺ influx in Epi myocytes promoted increased SR Ca²⁺ loading, leading to increased gain and CaT amplitude. Supporting this hypothesis is the finding in rat ventricular myocytes of increased SR Ca²⁺ load and CaT amplitude when APD is increased (42). Similarly, AP prolongation increases CaT amplitude in rabbit ventricular myocytes (38).

In contrast to our results, both rabbit and rat ventricular myocytes displayed reduced CaT amplitude when subjected to AP clamps using human AP templates of heart failure in which the notch was reduced and APD increased (11). The authors proposed that the smaller CaT amplitude resulted from a reduction in peak I_Ca,L that decreased synchrony of SR Ca²⁺ release. We cannot rule out dyssynchronous Ca²⁺ release in our experiments. However, it seems possible that its effect on CaT amplitude was mitigated by enhanced SR Ca²⁺ content mediated by the much greater AP prolongation during low pH_i (∼88%; Fig. 2D) compared with that of the heart failure AP templates (∼25%) used in the Cooper et al. study (11).

In summary, we have shown that, in contrast to low pH_o, intracellular acidosis has a marked inhibitory effect on rabbit ventricular I_to, perhaps mediated by Kv4.3. The repolarization effects of this inhibition were much greater in Epi than PM myocytes and included slowing of phase 1, attenuation of the notch, and elevation of the plateau. In addition, the low pH_i-induced prolongation of APD was greatest in Epi myocytes. In the intact heart this may have the undesirable effect of promoting transmural heterogeneity of repolarization under pathological conditions in which pH_i falls. Increased dispersion of repolarization has been shown to facilitate the occurrence of reentrant arrhythmias (26). By altering the trajectory of the AP waveform, low pH_i modulates calcium handling as reflected in a reduction in initial peak I_Ca,L, increased net Ca²⁺ influx via I_Ca,L, and increased CaT amplitude and duration. Thus, in addition to the direct action of [H⁺]_i on I_Ca,L (41), SR Ca²⁺ release and uptake (28), NCX (15), and diastolic Ca²⁺ (41), low pH_i also exerts a significant indirect effect on E-C coupling. This illustrates the striking diversity of mechanisms that contribute to the overall response of ventricular myocytes to acute increases in [H⁺]_i.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant R37 HL-042873 and the Nora Eccles Treadwell Foundation (to K. W. Spitzer) and an American Heart Association Postdoctoral Fellowship (to V. Garg).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

Author contributions: N.S., V.G., and K.W.S. conception and design of research; N.S., V.G., and K.W.S. performed experiments; N.S., V.G., and K.W.S. analyzed data; N.S., V.G., and K.W.S. interpreted results of experiments; N.S. and K.W.S. prepared figures; N.S. and K.W.S. drafted manuscript; N.S., V.G., and K.W.S. edited and revised manuscript; N.S., V.G., and K.W.S. approved final version of manuscript.