Block of Potassium Currents in Guinea Pig Ventricular Myocytes and Lengthening of Cardiac Repolarization in Man by the Histamine H1 Receptor Antagonist Diphenhydramine1

Materials and Methods

Isolated Heart Experiments

Experiments were performed in accordance with our institutional guidelines on animal use in research. Animals were housed and maintained in compliance with the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care.

Heart Isolation and Perfusion Technique.

Male Hartley guinea pigs (weight, 300–350 g; Charles River Laboratories, Montreal, Quebec, Canada) were anticoagulated by injection of heparin sodium (400 IU i.p.). Twenty minutes later, animals were anesthetized with sodium pentobarbital (25 mg · kg⁻¹ i.p., Somnotol; MTC Pharmaceuticals), and the hearts were rapidly extirpated and immersed in cold (4°C) Krebs-Henseleit buffer. Each heart was cannulated and retrogradely perfused via the aorta at a constant pressure equivalent to 100 cm of H₂O. To permit rapid exchange in perfusion solutions, a double warming-coil heart-perfusion system (Ealing Scientific Limited, St. Laurent, Quebec, Canada) and two parallel liquid columns were used. Krebs-Henseleit bicarbonate buffer, which contained 11.2 mM glucose, 4.7 mM KCl, 1.2 mM CaCl₂, 25 mM NaHCO₃, 118.5 mM NaCl, 2.5 mM MgSO₄, and 1.2 mM KH₂PO₄, was used as a perfusate. This solution was continually gassed with 95% oxygen plus 5% carbon dioxide (pH 7.4, 37°C) and filtered through a 5-μm cellulose acetate membrane to remove any particulate contaminants.

Electrophysiological Measurements.

Hearts were electrically stimulated (programmable stimulator model 5325; Medtronic) at a basic cycle length of 250 ms (4 Hz) at 3 times threshold via two silver electrodes implanted in the epicardium of the right ventricle. A monophasic action potential catheter (Langendorff probe model 225; EP Technologies, Inc.) was introduced in the left ventricle through the mitral valve and securely positioned to obtain visually adequate signals (amplitude >15 mV, stable phase 4). During the protocol, monophasic action potential signals were recorded on a computer for a duration of 6 s at 30-s intervals (digital sampling rate, 1 kHz) and stored on hard disk for analysis. Monophasic action potential duration was determined by analyzing all complete beats in the 6-s sample for monophasic action potential duration at 90% repolarization (MAPD₉₀). These values were averaged using a routine designed specifically for this purpose and incorporated into the computer program (CVRP92 Cardiovascular Research Partner, Datton System Enregistered). At least 10 complexes were used for each measurement.

Protocols.

A total of 51 hearts were perfused during a control period of 3.5 min to assess stability of monophasic action potential signal. Thereafter, perfusion solution was switched to one containing 10⁻⁵M DPH, 10⁻⁴ M or 10⁻³ M N-acetylprocainamide (NAPA), 10⁻⁴ M indapamide (IND), or a combination of 10⁻⁵ M DPH with 10⁻⁴ M NAPA, 10⁻³ M NAPA, or 10⁻⁴ M IND. After a 5-min exposure to drug(s), perfusion with Krebs-Henseleit buffer containing no drug was restarted to assess reversal of drug effects.

Statistical Analysis.

Only hearts with reversal of drug effects upon reperfusion with buffer containing no drug were included in the analysis. The last three values of MAPD₉₀ determined at baseline (2.5, 3.0, and 3.5 min) and during the drug infusion period (7.5, 8.0, and 8.5 min) were averaged and compared. Data were analyzed using a test of Repeated Measures Design. Statistical significance was set at P < .05.

Whole-Cell Voltage Clamp Experiments

Cell Preparation.

Experiments were performed on single ventricular myocytes obtained from adult guinea pig hearts by use of an enzymatic dissociation technique. All solutions used during the cell isolation procedure were oxygenated and maintained at 37°C. The hearts were mounted on a Langendorff apparatus and rinsed for 2 min with a calcium-free solution (solution A) containing 132 mM NaCl, 4.8 mM KCl, 10 mM HEPES, 1.2 mM MgCl₂, and 5 mM glucose; pH was adjusted to 7.35 with NaOH. Then the hearts were perfused with low-sodium high-potassium HEPES-buffered solution (solution B, 17 mM NaCl, 5.4 mM KCl, 10 mM HEPES, 1.2 mM MgCl₂, 5 mM glucose, and 128.3 mM potassium glutamate) for a period of 2 min. At the end of this period, perfusion of solution B containing collagenase (final concentration, 300 U/ml; Boehringer Mannheim, Indianapolis, IN) was started and continued until the system pressure dropped to 15 mm Hg (approximately 15 min). Hearts were reperfused with solution B for 3 min and then with a solution made of a mixture of solution B and solution A (85:15) containing 200 μM CaCl₂. Hearts were finally perfused with a solution made of 60% solution B and 40% solution A containing 500 μM CaCl₂. At this point, the ventricles were cut down and minced slightly. After filtration through 200-μm nylon mesh, the dispersed cells were resuspended in solution A containing 1.8 mM CaCl₂ and maintained at 30°C before use.

Extracellular and Drug Solutions

The external solution used to perfuse cells during recordings of potassium currents contained 145 mM NaCl, 1 mM MgCl₂, 0.1 mM CaCl₂, 10 mM HEPES, and 5 mM glucose. Nisoldipine (Bayer Leverkusen) 2 · 10⁻⁷ mol/liter was added to eliminate the slow calcium inward current (I_si). The pH of the extracellular solution was adjusted to 7.3 with KOH. During recording of calcium currents, the external solution contained 140 mM tetraethylammonium chloride, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, and 10 mM glucose; pH was adjusted to 7.3 with NaOH. DPH was added to extracellular solution to obtain desired final drug concentrations (3 · 10⁻⁶, 10⁻⁵, 3 · 10⁻⁵, 10⁻⁴, 3 · 10⁻⁴ or 10⁻³ M).

Patch Electrodes and Intracellular Solution.

Patch-clamp pipette electrodes were fabricated from glass capillary tubes (1.2 mm o.d.; Rednoti Technology, Inc.) on a microelectrode puller (model P-87; Sutter Instrument Co., Novato, CA) and heat-polished with a microforge. Pipettes had tip resistances of 3 to 5 MΩ when filled with the intracellular solution containing 500 mM KCl, 10 mM HEPES, 11 mM EGTA, 2 mM MgCl₂, 1 mM CaCl₂, 5 mM MgATP, and 5 mM K₂ATP during potassium current recordings and 140 mM CsCl, 10 mM EGTA, 10 mM HEPES, 3 mM MgATP, 0.4 mM MgGTP, and 10 mM glucose during calcium current recordings. The pH was adjusted to 7.2 with NaOH. A high intracellular concentration of K⁺ was used to reduce electrode tip size, preventing cell dialysis. This has been shown to reduce rundown of I_K without inducing important errors in response time and steady-state voltage (Balser et al., 1990).

Electrophysiological Measurements.

A small aliquot of dissociated cells was placed in a 0.5-ml chamber mounted on the stage of an inverted microscope (model CK2; Olympus). Cells were allowed to adhere to the coverslip at the bottom of the chamber and were superfused continuously with the external solution prewarmed (30°C) by a Peltier device (Medical Systems Corp., Great Neck, NY). In our experiments, complete replacement of external solution contained in the chamber was achieved within 2 to 3 min when the superfusion rate was 2 ml/min.

All currents were recorded in the whole-cell, voltage clamp configuration of the patch-clamp technique using an Axopatch-1D amplifier (Axon Instruments, Inc., Burlingame, CA). Voltage-clamp command pulses were generated by a 12-bit digital-to-analog converter (model TL/1; Axon Instruments, Inc.) controlled by the PCLAMP software package (version 4.05b; Axon Instruments, Inc.).

Protocols.

Rod-shaped cells with clear cross striations, resting potentials more negative than −75 mV, and stable delayed rectifier (I_K) and inward rectifier (I_K1) currents (as assessed during a baseline period of at least 4 min) were used. Effects of DPH on the rapidly (I_Kr) and slowly (I_Ks) activating components of I_K were studied in cells held at −40 mV (to inactivate I_Na) and depolarized by pulses lasting either 250 ms (I_K250) or 5000 ms (I_K5000). Test potentials of depolarizing pulses varied between −20 and +50 mV. I_K was measured from the peak amplitude of tail current obtained upon repolarization to −40 mV. A voltage ramp was used to obtain the current-voltage relation of the inward rectifier potassium current (I_K1). In this protocol, cells were held at −40 mV before their membrane potential was changed from 0 to −100 mV in 500 ms.

Slow inward calcium current (I_Ca-L) was elicited by depolarizing pulses (−40 to +40) lasting 250 ms from a holding potential of −50 mV. Peak amplitude was measured at all test potentials. In these experiments, nisoldipine was omitted in the bath solution and calcium concentration was fixed at 1.8 mM.

Data Storage and Analysis.

Currents were filtered at either 2 kHz (I_K250, I_K1, and I_Ca-Lprotocols) or 500 Hz (I_K5000 protocol) by a four-pole Bessel filter (−3 dB/octave). Currents were sampled at 4 kHz (I_K1), 2 kHz (I_K250 and I_Ca-L), and 400 Hz (I_K5000) by using of a 12-bit analog-to-digital converter (TL-1 DMA; Axon Instruments, Inc.) and stored on hard disk for subsequent analysis. Statistically significant block of I_K250 and I_K5000 was tested by randomized block design; significant difference of the I_K250 and I_K5000 block with different concentrations of DPH was tested by split-plot design and the difference between the block of I_Kr and I_Ks was assessed by a Student’st test. Statistically significant voltage dependence was tested by a conditional Hotelling’s t²test. In this analysis, a Shapiro-Wilk test was used to assess normality. The voltage-to-peak current, the peak outward current amplitude, and the inversion potential of the inward rectifier current (I_K1) were compared by Student’s t test. The level of statistical significance was set at P < .05.

Effects of DPH on Cardiac Repolarization in Humans

Administration of DPH to Healthy Volunteers.

A total of six male, 20 to 35 years old, healthy volunteers, received a single oral dose of DPH hydrochloride (Benadryl) after an overnight fast. The dose administered was either 50 or 100 mg (three subjects per dose). Electrocardiographic recordings (12-lead ECG) were obtained simultaneously at a paper speed of 25 and 50 mm/s using a MAC 15 recorder (Marquette Electronics, Inc.). Recordings were obtained before and at 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 210, 240, 270, 300, and 330 min after drug administration. Electrocardiographic measurements were made, in a blinded fashion, by an independent investigator using recently described Q-T measurement criterias (Cowan et al., 1988; Garson, 1993). Corrected Q-T for heart rate (Q-T_c) was determined using Bazett’s formula (Bazett, 1920).

Administration of DPH to Patients Undergoing Percutaneous Coronary Angioplasty (PTCA).

Twenty patients undergoing PTCA received a 100-mg oral dose of DPH hydrochloride (Benadryl) at bedtime the night preceding the intervention as a regular clinical procedure to prevent allergic reactions to contrast reagents. On the morning of PTCA, patients were administered another 50 mg of DPH hydrochloride 1 h before the scheduled procedure time. A first ECG recording was obtained before the administration of the evening dose of DPH; a second ECG was recorded at least 2 h after PTCA but in the interval of 3 to 4 h after the morning dose of DPH.

Results

Experiments performed in isolated, buffer-perfused guinea pig hearts demonstrated that DPH 10⁻⁵ M caused a significant increase in MAPD₉₀ in all hearts (n = 7) exposed to the drug (Fig.1A). Overall mean increase in MAPD₉₀ was 15 ± 6 ms (P < .05 versus baseline; Student’s paired t test); a typical example of monophasic action potential recorded at baseline and during perfusion of DPH 10⁻⁵ M is illustrated in Fig.1B.

Experiments performed with isolated guinea pig hearts also demonstrated that NAPA 10⁻⁴ M, NAPA 10⁻³ M, and IND 10⁻⁴ M all caused a significant increase in MAPD₉₀ (Fig.2A). Mean increases in MAPD₉₀ were 12 ± 7 (n = 10), 22 ± 10 (n = 8), and 18 ± 2 ms (n = 7), respectively (all P < .05 versus baseline). Combined perfusion of DPH 10⁻⁵ M with either NAPA 10⁻⁴ M or NAPA 10⁻³ M increased MAPD₉₀ by 17 ± 8 ms and 24 ± 4 ms, respectively (both P < .05 versus baseline; Fig. 2B). These increases were of a magnitude similar to that observed during the perfusion of either DPH alone (Fig.1A) or NAPA alone (Fig. 2A). In contrast, combined perfusion of DPH 10⁻⁵ M with IND 10⁻⁴ M was associated with pronounced prolongation of MAPD₉₀. Increase in MAPD₉₀observed under these conditions (41 ± 11 ms) was greater than that observed during DPH 10⁻⁵ M alone (15 ± 6 ms; P < .05), IND 10⁻⁴ M alone (18 ± 2 ms; P < .05), or their estimated additive effects (≈33 ms).

Effects of DPH on MAPD₉₀ when administered alone or in combination with other drugs were time-related and could be reversed upon removal of the drug(s). Figure3 illustrates that a stable signal was achieved in the first 3-min period during which hearts were perfused with buffer containing no drug. This period was long enough to assess stability of MAPD₉₀ measurement. Upon exposure to drug(s), a rapid increase in MAPD₉₀, which achieved maximal effect within a 5-min period, was noticed. Reperfusion with buffer containing no drug was associated with reversal of drug effects.

To elucidate the mechanism related to the effects of DPH on cardiac repolarization, experiments were conducted in isolated guinea pig ventricular myocytes using the patch-clamp technique. Figure4 illustrates outward membrane currents elicited by a 250-ms long pulse (I_K250) from holding potential (−40 mV) to various test potentials. Elicited currents exhibited time-dependent deactivation characteristics upon repolarization to −40 mV. Superfusion of the cell with DPH 3 · 10⁻⁵ M significantly reduced I_K250 time-dependent activating and deactivating currents. A progressive but near complete recovery of the tail current was observed after removal of the drug. A total number of 27 cells was exposed to DPH 3 · 10⁻⁶ to 10⁻³ M. In all of these cells, I_K250 tail current decreased in a very reproducible manner. Decrease in I_K250 was also concentration-dependent with an estimated IC₅₀ of 3 · 10⁻⁵ M (Fig.5).

Figure 6 illustrates recordings of currents elicited by 5000-ms long pulses (I_K5000) under control conditions in the presence of DPH 10⁻⁴ M and after removal of the drug. Activating currents were elicited by various test potentials, whereas deactivating tail currents were recorded after repolarization to −40 mV. DPH 10⁻⁴ M reduced, in a reversible manner, time-dependent activating and tail currents. Decrease in I_K5000 was reproducibly observed in a total of 32 cells exposed to DPH 3 · 10⁻⁶ to 10⁻³ M with an estimated IC₅₀ of 1.3 · 10⁻⁴ M (Fig. 7).

We have also performed experiments in the presence of dofetilide 1 · 10⁻⁷ M to measure the block of I_Ks in the absence of I_Kr. Figure 8 illustrates that a ∼50% decrease in tail current is observed upon exposure of the cell to DPH 10⁻⁴ M, i.e., close to IC₅₀ for block of I_Ks.

Voltage-dependent block of I_K250 and I_K5000 was assessed at IC₅₀for block of I_Kr and I_Ks, respectively. Decrease in I_K250 tail current reached 60% for test potentials of −20 and −10 mV but only 39% after depolarization to more positive potentials (+50 mV; P< .05; Fig. 9A). This is most likely explained by the activation of I_Ks at high depolarizing potentials and by the higher IC₅₀for block of this component by DPH. Tail current of I_K5000 was also decreased in a voltage-dependent manner by DPH (P < .05; Fig. 9B).

At concentrations varying from 3 · 10⁻⁵ to 3 · 10⁻⁴ M, DPH did not alter the time-independent background current (mostly I_K1) elicited by a ramp of voltages (0 to −100 mV in 500 ms). DPH had no effects on reversal potential, voltage of peak current, and peak outward current amplitude (Table 1). Finally, DPH 3 · 10⁻⁵ M (n = 4) did not alter the slow inward calcium current I_Ca-L.

Administration of DPH to healthy volunteers appeared to cause a concentration-dependent prolongation of the Q-T_cinterval (Table 2). In subjects exposed to 50 mg of the drug, maximal prolongation observed in Q-T_c varied between 9 and 14 ms, whereas maximal prolongation reached 23 to 38 ms in subjects exposed to 100 mg of DPH. Changes in Q-T_c were time-related and for all subjects studied, maximal increase in Q-T_coccurred between 3 and 4 h after drug administration.

Administration of DPH to patients undergoing PTCA was also associated with an overall increase (14 ± 26 ms; P < .05 versus baseline) in Q-T_c (Table3). In seven patients, the increase in Q-T_c was greater than 20 ms (23–64 ms), whereas a decrease in Q-T_c of more than 20 ms was observed in two patients (25 and 34 ms).

Discussion

Results obtained in this study demonstrate that the classic histamine H1 receptor antagonist DPH blocks, at relatively high concentrations, the rapid component of the delayed rectifier, I_Kr. The extent of lengthening of the Q-T_c interval observed after administration of the drug to healthy volunteers suggests that prolongation of cardiac repolarization due to DPH should be of limited clinical relevance in most patients. However, unheralded toxicity could be expected in some patients due to various pathological conditions (ischemia, arrhythmia), concomitant drug administration, limited clearance capability, or overdose administration of DPH.

At concentrations used in our study, NAPA and IND are selective blockers of I_Kr and I_Ks, respectively (Turgeon et al., 1990, 1994). Consequently, exposure of isolated guinea pig hearts to these drugs was associated with prolongation of cardiac repolarization as assessed by MAPD₉₀. Effects of DPH on MAPD₉₀ were compared to those of these selective blockers. As well, we determined MAPD₉₀ during the combined exposure of hearts to DPH and either NAPA or IND. Our results demonstrated that at a concentration of 10⁻⁵ M, DPH prolonged MAPD₉₀ to an extent similar to that observed with either NAPA 10⁻⁴ M or IND 10⁻⁴ M. However, during combined perfusion of DPH 10⁻⁵ M and IND 10⁻⁴M, prolongation of MAPD₉₀ was greater than that expected by additive effects of the drugs. Results obtained using isolated myocytes and the patch-clamp technique lead us to propose that combined block of I_Kr and I_Ks by DPH and IND could explain this increase in drug effects.

In contrast, combined perfusion of DPH and NAPA resulted in MAPD₉₀ prolongation to an extent similar to that observed during perfusion of either drug alone. Previous studies byRubart et al. (1993) demonstrated that the combined administration of specific I_Kr blockers, namely, erythromycin (Daleau et al., 1995; Antzelevitch et al., 1996) and dofetilide (Carmeliet, 1992; Jurkiewicz and Sanguinetti, 1993), was associated with a decrease in action potential prolongation relative to that observed during the administration of either drug alone.

In our experiments with isolated guinea pig ventricular myocytes, composition of the extracellular solution used to superfuse myocytes (nisoldipine 2 · 10⁻⁷ M and omission of calcium) allowed us to better separate the components of I_K (Sanguinetti and Jurkiewicz, 1992). I_K obtained for low depolarizing potentials (−20 to 0 mV) of relatively short duration (250 ms) presented characteristics of I_Kr: rapid activation that reaches a quasi steady-state level after a few hundred milliseconds and fast inactivation properties clearly observed by comparing activating and tail current amplitudes at −10 and 0 mV. On the other hand, I_K obtained at voltage tests ≥+10 mV of relatively long duration (5000 ms) presented characteristics of I_Ks: a slowly activating time-dependent current with an early sigmoidal shape that does not reach a steady-state level even after 5 s of depolarization (Sanguinetti and Jurkiewicz, 1990). Results obtained demonstrated that DPH reduces I_K tail current mainly during short pulses to low depolarizing voltages. In fact, DPH produces greater inhibition of the I_K250 tail current after pulses to −20, −10, and 0 mV compared to that observed for voltage steps ≥+20 mV. DPH inhibits I_K during activation characteristics typical of I_Kr. When the test depolarization was prolonged to 5000 ms, the decrease in I_K tail current was also greater for pulses to low depolarizing voltages (0, +10, and +20 mV) compared to high positive voltages (+30, +40, or +50 mV). In addition, inhibition of I_K by DPH was time-dependent: the longer the pulse was, the smaller was the inhibition. Characteristics of time-dependent block of I_K by DPH can be explained by relative proportions of I_Kr and I_Ksduring short and long depolarizing pulses, because I_Ks requires a longer time than I_Kr to activate. Thus, voltage dependence and time dependence of inhibition observed with DPH are consistent with a selective block of I_Kr.

Results obtained with guinea pigs using in vitro and ex vivo models were supported by data obtained in healthy volunteers and patients exposed to DPH. In fact, prolongation of cardiac repolarization as assessed by Q-T_c measurements was observed in all healthy volunteers and in 14 of 20 patients exposed to the drug and undergoing PTCA. Prolongation of cardiac repolarization was limited as predicted by the IC₅₀ for block of I_Kr and mean plasma concentrations obtained after administration of DPH at usual dosages (7 · 10⁻⁷ M; Simons et al., 1990). Although prolongation of the Q-T_c was usually minimal in most of the subjects, a clinically significant lengthening of cardiac repolarization (>20 ms increase in the Q-T_cinterval) was observed in 7 of 20 patients. Data obtained with our ex vivo model would suggest that concomitant drug therapy with DPH in these patients could explain a greater than expected increase in their Q-T interval.

Intersubject variability in drug metabolism activity may also account for nonuniform susceptibility to drug toxicity. Recent data from our laboratory have indicated that DPH inhibits CYP2D6 and that this enzyme may also be involved in its metabolism (Hamelin et al., 1998). Consequently, decreased capability in the clearance of DPH, i.e., increased plasma concentrations at the same dosage, could partially explain intersubject variability in drug action. Whether metabolites participate to DPH action and whether formation of these metabolites is favored or decreased in subjects with extensive or poor CYP2D6 activities remain to be demonstrated. Data from Yasuda et al. (1994)suggested that active metabolites of other first generation histamine H1 receptor antagonists such as chlorpheniramine are responsible for drug actions.

Of the histamine H1 receptor antagonists known to cause prolongation of cardiac repolarization and even torsades de pointes, possibly the best studied drug is terfenadine. Studies with isolated cardiac myocytes and isolated hearts demonstrated that terfenadine lengthens cardiac repolarization through a quinidine-like block of I_K (Woosley et al., 1993). This effect of terfenadine is observed mainly at high concentrations of the drug (10⁻⁷ M) reached during combined treatment with CYP3A4 inhibitors such as ketoconazole and macrolide antibiotics (Kuang et al., 1994; Salata et al., 1995). Previous studies in isolated hearts had also suggested that DPH is able to prolong cardiac repolarization, although the drug was less potent than terfenadine (Woosley, 1996). As well, Zareba et al. (1997) recently reported lengthening of the Q-T interval in patients administered an overdose of DPH.

In summary, results obtained in this study indicate that DPH selectively blocks the rapid component of the delayed rectifier potassium current (I_Kr) of isolated cardiac myocytes. In isolated, buffer-perfused guinea pig hearts, prolongation of monophasic action potential was consistent with demonstrated block of I_Kr. Administration of clinically relevant doses of the drug to humans slightly prolonged Q-T_c interval. However, unexpected cardiac toxicity may be observed at higher concentrations of the drug or during combined drug therapy.