RSD1000: A Novel Antiarrhythmic Agent with Increased Potency under Acidic and High-Potassium Conditions

Materials and Methods

In Vivo Studies

All experiments (approved by The Animal Care Committee of the University of British Columbia) were conducted on male Sprague-Dawley rats weighing 200 to 300 g. Rats were anesthetized with pentobarbitone (60 mg/kg, i.p.). In intact rats, an endotracheal tube (14 Jelco IV catheter) was inserted, the left carotid artery was cannulated for blood pressure recording, and right jugular vein cannulated for drug administration (Harvard Syringe pump, model 55–2222). ECGs were recorded from needle electrodes placed in an approximate lead V2 configuration. Signals were recorded on a Grass polygraph (model 79D) at a standard chart speed of 100 mm/s. Using a Harvard Miniature ventilator pump (model 50–1700) artificial ventilation was set at 10 ml/kg, 60 times a minute. Body temperature was maintained at 36 to 38°C.

Ischemia-Induced Arrhythmias.

Ischemic arrhythmias were induced by occlusion of the left coronary artery as previously described (Barrett et al., 1995). A specially constructed occluder, consisting of a polypropylene thread (5–0, Ethicon 8720H) inserted into a polyethylene guide (PE-10), was loosely placed around the left coronary artery at the level just below its first bifurcation. Rats were allowed 30 min to recover from surgery before random and blind drug treatment. Arterial blood samples were taken before and after coronary artery occlusion for determination of serum potassium concentrations using a potassium ion selective electrode (Ionetics Potassium Analyzer, Ionetics, CA, USA). After 5 min of drug infusion, the occluder was permanently tightened and drug infusion maintained. All arrhythmias were recorded for 15 min postocclusion. The arrhythmic history of each animal was expressed as an arrhythmia score (AS) (Curtis and Walker, 1988) following the guidelines outlined in the Lambeth Conventions (Walker et al., 1988). At the end of the experiment, hearts were excised and perfused with piperazine-N,N′-bis[2-ethanesulfonic acid] (PIPES) buffer containing cardiogreen dye (0.2 mg/liter Fast Green FCF) to differentiate between underperfused (occluded zone) from perfused (green) tissue. The former region was excised and weighed to give the size of the occluded zone as a percentage of the total ventricular mass (%OZ).

Electrically Induced Arrhythmias.

The actions of RSD1000 on resistance to electrical stimulation and induction of arrhythmias was assessed in vivo in normal hearts according to the method of Walker and Beatch (1988). Stimulating electrodes were implanted by a transthoracic route in the apical region of the left ventricle and square wave stimulation (at 7 Hz; Grass model SD9 stimulator) was used to determine threshold current (iT) and pulse width (tT) for induction of extrasystoles, threshold current for induction of ventricular fibrillo-flutter (VFt, μA) at 50 Hz, and effective refractory period (ERP, ms) at 7 Hz. Before drug infusion, each variable was measured three times every 5 min until consistent values were obtained. Animals were randomly allocated to vehicle or RSD1000 infusion (1, 2, 4, 8, and 16 μmol/kg/min). Drug infusion was continuous for the duration of the experiment with successive incremental doubling of the previous infusion with each infusion level lasting 5 min. At the end of the third minute, electrical stimulation endpoint measurements (in duplicate) were made; this took 2 min. Because there was no definable maximal response for any of the above measures, ED₅₀ values were unobtainable. Therefore, doses producing a 50% change from predrug values were interpolated from the dose-response data and were expressed as D₅₀% values.

In Vitro Studies

Isolated Rat Hearts.

The actions of RSD1000 in isolated rat hearts were studied using a modified Langendorff perfusion apparatus (Curtis et al., 1986). “Normal” PIPES buffer was of the following composition: 153 mM NaCl, 3.4 mM KCl, 1.18 mM MgSO₄·7H₂0, 11.1 mMd-glucose, 2.52 mM CaCl₂·2H₂O, and 14.34 mM PIPES. The buffer was titrated to pH 7.4 with NaOH and aerated with oxygen. Acid (pH_o = 6.4) and raised [K⁺]_o buffer was adjusted by titriating with HCl and adding 10.8 mM KCl in place of 3.4 mM KCl.

Rats were overdosed with pentobarbitone (70 mg/kg, i.p. plus heparin, 1000 U). Excised hearts were washed with ice-cold buffer before being perfused at 100 mm Hg and 37°C. A noncompliant balloon, inserted in the left ventricle at an end diastolic pressure of 5 mm Hg, was used to measure left ventricular pressure. Epicardial ECGs were recorded using two silver ball electrodes attached to separate 1-cm circular disks of filter paper. Hearts were stabilized for 20 min in normal buffer before being randomized to normal or “ischemic” buffer containing vehicle or RSD1000. The concentration range of RSD1000 tested was 0.01 to 300 μM. Each concentration was perfused for 3 min before heart rate, ECG variables, and ventricular pressure were measured.

Isolated Rat Ventricular Myocytes.

The methods used to prepare dissociated rat ventricular myocytes generally followed those previously described (Mitra and Morad, 1985); the specific procedures employed have also been previously detailed (McLarnon and Xu, 1995). A constant-flow Langendorff system was used during the isolation of cells with oxygenated Tyrode’s solution containing: 133.5 mM NaCl, 4 mM KCl, 1.2 mM MgCl₂, 1.2 mM NaH₂PO₄, 10 mMN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), and 11 mM glucose, and pH was adjusted to 7.3 with 1 mM NaOH. Following 15 to 20 min of enzyme treatment (0.07% Type II collagenase, Worthington Biochemical), Tyrode’s solution (with 25 μM Ca²⁺) was reapplied with gentle agitation of the digested tissue. Cell suspensions were centrifuged and resuspended in fresh Tyrode’s solution. The cells were stored at room temperature between intervals of washing with successively increased Ca²⁺concentrations (final concentration = 1.8 mM). The morphology of the cells was rod-shaped and quiescent and their percentage yield was in the range of 70%.

Electrophysiological Studies of Single Rat Ventricular Myocytes.

The procedures used in this laboratory for the recording of macroscopic currents from isolated rat ventricular myocytes have been described previously (McLarnon and Xu, 1995; 1997). In the present study, whole-cell transient outward K⁺(I_TO), inward Na⁺(I_Na) and inward calcium (I_Ca) currents were recorded. The micropipettes were made from Corning 7052 glass (A-M Systems, Everett, WA) with resistance values between 2 to 4 MΩ. An axopatch amplifier (model 200A, Axon Instruments, Foster City, CA) was used to record the currents with the low-pass filter set at 1 or 2 Khz. Capacitive current and series resistance were compensated using analog circuitry of the amplifier. Holding potentials were from −70 to −100 mV and voltage clamp protocols were operated by computer using pClamp 6 (Axon Instruments). I_TO was activated with a depolarizing step to +60 mV from −70 mV (a holding potential of −80 mV was also used in some experiments, see Results section). I_Na was recorded with a series of depolarizing steps to a potential of −20 mV following an initial prepulse to −140 mV from holding potential to remove resting inactivation. Concentration-response curves were plotted for the effects of RSD1000 on the time courses of decay of I_TO and the amplitudes of I_Na. Use-dependent block of I_Na by RSD1000 was investigated with the application of depolarizing steps to −20 mV from a holding potential of −100 mV. The protocol consisted of a series of 20 pulses (pulse durations of 20 ms) applied at a frequency of 20 Hz and the data were analyzed by plotting normalized current for each of the 20 episodes. I_Ca was recorded following a single depolarization step to +20 mV from a holding potential of −70 mV. Statistical significance was determined using Student’s ttest or two-way ANOVA and all data were recorded at room temperature (21–24°C).

Experimental Solutions.

The bath solution used to record I_TO was a modified Tyrode’s solution and contained: 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.2 mM CdCl₂, 0.5 mM MgCl₂, 5 mM glucose, 10 mM HEPES, and pH was adjusted to 7.3 (or pH 6.4) with NaOH. Cadmium and tetrodotoxin (5 μM) were added to the bath solution to suppress I_Ca and I_Na, respectively. The patch pipette solution contained: 120 mM KCl, 0.15 mM CaCl₂, 6 mM MgCl₂, 5 mM EGTA, 5 mM Na₂-ATP, and 10 mM HEPES; pH was adjusted to 7.3 with KOH.

The bath solution used to study I_Na contained 5.4 mM CsCl (to replace 5.4 mM KCl) and 50 mM NaCl with 87 mM Tris (to replace 137 mM NaCl). The pH of the bath solution was titrated to 7.3 or 6.4 with HCl. 4-Aminopyridine at 5 mM was also included in the bath solution to block I_TO. The pipette solution contained: 10 mM NaCl, 120 mM CsCl, 12 mM EGTA, 10 mM TES, 1 mM MgCl₂, and 5 mM Na₂-ATP; pH was adjusted to 7.3 with KOH.

Experiments on I_Ca used the following ion composition in the bath solution: 137 mM Tris, 5.5 mM CaCl₂, 1 mM MgCl₂, 20 mM CsCl, and 5.5 mM glucose (5.5); pH was adjusted to 7.3 with CsOH. I_Na and I_TO currents were suppressed with external addition of 5 μM tetrodotoxin and 5 mM 4-aminopyridine. The pipette solution used: 125 mM CsCl, 5 mM Mg-ATP, 15 mM EGTA, 20 mM TEACl, and 10 mM HEPES; pH was adjusted to 7.3 with CsOH.

Drugs

RSD1000 was synthesized by Nortran Pharmaceuticals Inc., Vancouver, British Columbia, Canada. The pK_a of RSD1000 was determined using by the equivalence point method. pH was plotted versus volume (milliliters) of base added (HEPES; pK_a = 7.35) and the equivalence point (temp. = 25°C) determined. RSD1000 was made fresh before each experiment and was soluble in a solvent consisting of 22% ethanol and 78% distilled water.

Statistical Analysis

Results are presented as the mean ± S.E.M. (vertical lines). When comparing pre- with postdrug values, Student’st test was used (P < .05). Statistical analysis between vehicle and treated groups was performed by repeated measures ANOVA followed by Tukey’s t test (P < .05) or Dunnett’s test (P < .05 or P < .001). In cases where mortality was the endpoint, statistical analysis was performed using the χ-square test with P < .05. The effects of pH were evaluated by comparing drug changes in acid and normal pH using Student’s pairedt test.

Results

Effects of RSD1000 on Hemodynamic, ECG Variables and Ischemia-Induced Arrhythmias

Hemodynamic and ECG variables were measured 5 min after beginning treatment, before coronary artery occlusion. RSD1000 dose dependently decreased blood pressure, heart rate, and prolonged both P–R and Q–T intervals (Tables 1 and2). Statistically significant effects occurred at 1 μmol/kg/min for lowering blood pressure and 4 μmol/kg/min for increasing P–R and Q–T intervals. Although mean blood pressure was decreased with increasing dose, the mean pulse width was unchanged by RSD1000. In the sham occlusion experiment, RSD1000 at 8 μmol/kg/min demonstrated that the hypotensive action of RSD1000 was not progressive during the duration of the experiment.

The antiarrhythmic action of RSD1000 against ischemia-induced arrhythmias in intact rats is summarized for all arrhythmias in Table3 and is summarized as a reduction in mean AS in Fig. 2. The incidence of premature ventricular contractions (PVC), ventricular tachycardia (VT), and ventricular fibrillation (VF) occurring after coronary artery ligation were decreased in RSD1000-treated animals, relative to control, in a dose-dependent manner. Antiarrhythmic data points were best fit using the equation,Equation 1where x specifies concentration of RSD1000,y denotes AS, a is ED₅₀, and h is the Hill coefficient. The response, y, was factored by 6.25 to scale the response to 100% efficacy. The ED₅₀ for antiarrhythmic activity was estimated to be 2.5 ± 0.1 μmol/kg/min (h = −2.8) with complete protection against ventricular tachycardia and fibrillation occurring at 8 μmol/kg/min (Table 3).

Effects on Electrically Induced Arrhythmias

To reveal the possible ion channel blocking actions of RSD1000 in vivo and its effects on arrhythmias in normal myocardium, RSD1000 was tested against arrhythmias produced by electrical stimulation of the left ventricle in intact rats. Figure 3shows that RSD1000 at infusion levels greater than those in Fig. 2increased threshold currents for both electrical induction of VFt and extrasystoles (iT) and also increased ERP in a dose-related manner. Data points were fitted to lines using nonlinear equations and their D₅₀% values were estimated from seven determinations. D₅₀% values for VFt, iT, and ERP were estimated to be 15 ± 3.2, 11 ± 1.4, and 7.8 ± 0.9 μmol/kg/min, respectively, demonstrating that at higher doses RSD1000 has actions in nonischemic rat myocardium, possibly related to block of Na⁺ and K⁺currents.

Effects of RSD1000 in Normal and Simulated Ischemia Buffers

The actions of RSD1000 in normal buffer and a simulated ischemic buffer were investigated in isolated rat hearts. Changes induced by RSD1000 were expressed as percentage of changes from pretreatment and plotted in Fig. 4 in terms of P–R interval and QRS duration. Maximal responses for heart rate, P–R and QRS could not be obtained, because atrioventricular block occurred at the highest concentrations of RSD1000; atrioventricular block occurred at an average RSD1000 concentration of 1 μM (n = 6) in ischemic and 300 μM (n = 6) in normal buffers. RSD1000 produced a concentration-dependent decrease in heart rate and an increase in P–R interval and QRS duration. Drug effects were more pronounced on heart rate (results not shown) and P–R interval. In addition, a slight decrease in ventricular pressure was observed under both buffer conditions (results not shown). Data points were fitted to lines using nonlinear equations and their C₂₅% values were estimated from seven determinations. We compared the relative potency of RSD1000 in each condition and estimated the concentrations required to produce a 25% change from predrug values (C₂₅%). RSD1000 was approximately 40 times more potent in the ischemic buffer. For example, the C₂₅% values in ischemic and normal buffers for P–R interval increases were 0.8 ± 0.3 μM and 34 ± 7 μM, respectively (P < .05).

Effects of RSD1000 in Single Ventricular Myocytes

Inward Sodium Current (I_Na)

Inward sodium currents were elicited from a resting potential of −70 mV by initiating a hyperpolarizing prepulse to −140 mV (to remove inactivation) and depolarizing to −20 mV (see pulse protocol, Fig.5). Original traces in Fig. 5A illustrate the control currents (bottom traces) and the effects of 2 μM RSD1000 on I_Na currents at pH 7.3 and 6.4 (top traces). Concentration responses are shown in Fig. 5B for n= 4 cells with bath solutions at pH 7.3 or 6.4. The lines are best fits using eq. 1 (see above) and the EC₅₀ values were estimated to be 2.9 ± 0.3 μM (h = −1.1) at pH 7.3 and 0.8 ± 0.1 μM (h = −0.8) at pH 6.4. These results show that the blocking action of RSD1000 on I_Na was significantly enhanced (P < .05) under external acid conditions.

The current-voltage (I-V) relationship of RSD1000 on I_Na was investigated by eliciting a series of depolarizing steps from −100 to +30 mV at a frequency of 0.5 Hz (V_H = −100 mV). Figure6 shows peak I_Naamplitudes as a function of depolarizing potentials in the absence (closed symbols) and presence (open symbols) of 5 μM RSD1000 at both pH 7.3 and 6.4. The data are shown with least square best fit of the Boltzman equation in the form:Equation 2where I_Na is the maximal sodium current,G_max is the maximal channel conductance, E_rev is the membrane potential at which the current is zero, V′ is the membrane potential at which one half maximal activation occurs, k is the slope factor for the voltage dependence of activation. In control pH 7.3 and 6.4, the threshold for I_Na activation was −60 mV with peak I_Na amplitudes of approximately 16 to 18 nA between −40 and −30 mV. At pH 7.3, RSD1000 (n = 4) decreased the peak I_Naamplitude without changes in the threshold and peak potentials (Fig.6A). In contrast, RSD1000 (n = 4) significantly suppressed peak I_Na (P < .05) and its activation to more positive potentials (Fig. 6B). Thus, voltage-dependent actions of RSD1000 to more positive potentials appear to be present at pH 6.4 with no dependence at pH 7.3. The parameters for the best fit in each case were: pH 7.3 control,G_max = 77.6 ± 5.9 nanoSiemens (nS), E_rev = 21.1 ± 2.4 mV, V′ = −46.4 ± 0.9 mV, and k = 3.3 ± 0.6; pH 7.3 and 5 μM RSD1000,G_max = 64.1 ± 9.1 nS,E_rev = 21.3 ± 3.9 mV,V′ = −43.4 ± 2.4 mV, and k = 5.9 ± 1.6. Under pH 6.4 conditions, the parameters were: pH 6.4 control,G_max = 83.3 ± 3.7 nS,E_rev = 25.5 ± 1.3 mV,V′ = −43.8 ± 0.6 mV, and k = 5.3 ± 0.4; pH 6.4 and 5 μM RSD1000,G_max = 24.6 ± 3.5 nS,E_rev = 47.7 ± 6.6 mV,V′ = −35.2 ± 1.3 mV, and k = 5.4 ± 0.8.

Use-dependent inhibition of I_Na was studied using a series of 20 depolarizing steps to 20 mV from a holding potential of 100 mV applied at a frequency of 20 Hz (Fig.7). We first determined whether any use-dependent effects occurred in the absence of RSD1000. Our results showed that there was no change in I_Na amplitude during the sequence of pulses for either pH 7.3 or 6.4 (data not shown). Original sample traces of I_Na currents associated with the 1st, 2nd, 5th, 10th, and 20th pulses are shown in Fig. 7A for both pH values in the presence of 5 μM RSD1000. At pH 7.3 there was a small decrease in I_Na amplitude with increasing pulses. In this cell, the amplitude of the last current was 70% relative to the initial current evoked in the series. Overall, at pH 7.3 the amplitude of the 20th evoked I_Na was 71 ± 8% of the initial current (n = 4). In contrast, use-dependent inhibition of I_Na by RSD1000 was very prominent when external pH was 6.4. In Fig. 7A, the amplitude of the final current was 36% of the initial amplitude. Overall, at pH 6.4 the amplitude of the last I_Nawas 37 ± 5% relative to the initial current of the series (n = 4). A plot of the normalized current for each of the 20 steps is presented in Fig. 7B for both pH conditions in the absence (open symbols) and presence (closed symbols) of 5 μM RSD1000. These results show that use-dependent block of I_Na was significantly enhanced when myocytes were exposed to an external solution with pH 6.4 compared with pH 7.3 (P < .05).

Transient Outward Current (I_TO).

In Fig.8A, sample traces of I_TO are shown following depolarizing steps to +60 mV from a holding level of −70 mV with pH at 7.3. The effects of RSD1000, applied at concentrations of 2 (middle trace) and 30 μM (bottom trace) on I_TO are shown. In this cell, the time constant of current decay (τ) was diminished to 70% (with 2 μM RSD1000) and 15% (with 30 μM RSD1000) of control value. The same experiments were also repeated at pH 6.4 in the absence or presence of RSD1000. The sample traces at pH 6.4 (Fig. 8A) relative to those shown at pH 7.3 illustrate an equipotent suppression of I_TO at concentrations of 2 μM (middle trace) and 30 μM (bottom trace) RSD1000. Concentration-response curves for RSD1000 actions on inactivation time course of I_TO are shown in Fig. 8B for both pH values (n = 5 cells). Overall, the EC₅₀was 2.8 ± 0.1 μM at pH 7.3 and 3.3 ± 0.4 μM at pH 6.4 and were not significantly different (P > .05). There was also no significant difference between potency of RSD1000 at the two different pH values if the area under the curve was used as an index of effect (data not shown). A previous study on the benzopyran compound, terikalant, also showed no difference in potency if the area under the curve or τ was used as a measure of response (McLarnon and Xu, 1995).

In the experiments described above the holding potential was −70 mV and it was possible that at this level not all I_TO was available for activation. To study this point we also carried out additional experiments using a concentration of RSD1000 (10 μM) with V_H = −80 mV (external pH at 7.3). In six cells, RSD1000 reduced the decay time constant of I_TO to 66 ± 6% of control (data not shown). This result can be compared with a reduction of 69 ± 3% found at V_H = −70 mV and indicates that there was no significant difference in the effects of RSD1000 to τ at holding potentials of −70 or −80 mV (P > .05).

Inward Calcium Current (I_Ca).

We also investigated the actions of RSD1000 on I_Ca currents in rat myocytes. Figure 9 shows the original current traces before and after superfusion of 30 μM RSD1000. This concentration was chosen because at this level RSD1000 strongly inhibited both I_Na and I_TO(Figs. 5 and 8). Using a high Ca²⁺-containing solution (see Materials and Methods), inward Ca²⁺ currents with amplitudes between 2 to 2.5 nA were recorded with 60-ms depolarizing steps to +30 mV from a holding potential of −70 mV. RSD1000 (30 μM) showed no evident effect to alter either the amplitudes or the time courses of calcium currents (n = 6 cells).

Discussion

The results of this study showed that RSD1000 provided almost complete protection against arrhythmias due to regional myocardial ischemia following coronary artery occlusion in rats. Furthermore, such protection occurred at doses that were lower than those that protected against electrically induced arrhythmias or depressed blood pressure. In vivo studies in normal hearts suggested that at higher doses RSD1000 blocked sodium currents (in a frequency-dependent manner) as well as potassium currents. This view was confirmed in studies with isolated myocytes in which RSD1000 was shown as a potent inhibitor of both I_Na and I_TO. In isolated hearts, evidence was obtained, at least for sodium channel blockade, that RSD1000 was more potent in conditions of simulated ischemia. This observation was also confirmed in isolated myocytes where the compound was more potent as a sodium current blocker at pH 6.4.

It was unlikely that the depressant effects on blood pressure and heart rate determined in vivo (see Tables 1-3) were due to inhibitory actions on calcium channels because the in vitro measurements showed that a high concentration of RSD1000 (30 μM) had no effects on I_Ca (Fig. 9). Instead, inhibition of I_TO currents may prolong refractoriness in sinoatrial pacemaker cells (Dukes and Morad, 1991) and partly account for the negative chronotropic effect of RSD1000. In addition, the sodium-blocking component of RSD1000 may also reduce heart rate by increasing the threshold for pacemaker discharge and depress blood pressure by producing negative inotropy with or without additional influences on the peripheral circulation.

RSD1000 was synthesized to be selective for ischemic myocardium by minimizing the concentration of the cationic species in nonischemic and, possibly, extra-cardiac tissues. This was accomplished by minimizing its degree of ionization at physiological pH relative to the raised extracellular [H⁺] during acute myocardial ischemia. The N-morpholino group of RSD1000 (Fig.1) is the tertiary nitrogen that contributes to the overall pK_a value of 6.1. In acid pH (6.4), the majority of RSD1000 is protonated, whereas at pH 7.3 only about 5% is charged. In acutely ischemic myocardial tissue, this should result in a local increase in the concentration of the protonated form of RSD1000. Determination of tissue levels of RSD1000 in ischemic versus nonischemic hearts was not performed in this study. It should be noted, however, that sufficient infusion time was given to achieve a “pseudo” steady-state level of RSD1000 in the heart before coronary artery ligation. In the period following ligation, compound levels in the uninvolved zone should continue to increase (up to a maximum), whereas those in the involved zone should remain unchanged or, alternatively, “trapped” because there was no apparent blood flow for drug removal. The low incidence of collateral flow present in the rat heart (Maxwell et al., 1987) would lessen the transfer of the compound from the left to right bed. On the contrary, the involved zone could receive RSD1000 via collateral flow from the right to left bed. In any case, lower concentrations were required in the isolated rat tissues under simulated ischemic or extracellular pH 6.4 conditions to produce equipotent effects on ECG intervals (Fig. 4) and reduction in peak I_Na, respectively. Any levels present in the involved zone following coronary ligation would likely be more than adequate to produce its effects.

Previous studies, using the same ischemic-arrhythmia model as for this study, failed to show that flecainide (ED₅₀ = 5.4 ± 0.8 μmol/kg/min; Barrett et al., 1995) or quinidine (ED₅₀ = 2.8 ± 0.7 μmol/kg/min; Barrett et al., 1995) provide antiarrhythmic protection in the manner that was dose dependent, pharmacologically tolerable, and selective for ischemic myocardium (for comparison, see Barrett et al., 1995). However, lidocaine was more effective as an antiarrhythmic (ED₅₀ = 5.7 ± 1.8 μmol/kg/min; Barrett et al., 1995), partly because of its frequency- and depolarization-dependence (Wendt et al., 1993), but only at doses that caused severe hypotension and convulsions in conscious rats (Barrett et al., 1995). Although the above findings with quinidine, lidocaine, and flecainide were obtained in rats, similar observations have been reported in other species in the setting of ischemia-infarction (Kupersmith, 1979; Carson et al., 1986; Aupetit et al., 1993) and, indeed, in clinical settings (Pentecost et al., 1981; Velebit et al., 1982; Echt et al., 1991; Morganroth and Goin, 1991).

RSD1000 is different from the classical antiarrhythmics because it has a unique pharmacological profile in terms of producing sodium current blockade that is pH-, ischemia- and frequency-dependent, while its potassium current blocking actions are still maintained at pH 6.4 conditions. This is not simply because RSD1000 is a lidocaine-like agent, because RSD1000, unlike lidocaine, does inhibit potassium channel(s). In addition, RSD1000 was much more effective as an antiarrhythmic than quinidine, agents that produce moderate frequency-dependent sodium current and I_TOblockade. Moreover, antiarrhythmic activity primarily via selective inhibition of I_TO has been shown to be associated with significant APD (or Q–T interval) prolongation (Beatch et al., 1991). This does not appear to be the case with RSD1000 and the above comparisons further imply that RSD1000 must have some special attributes to explain its preferred actions against ischemia-induced arrhythmias.

The pH-dependent action of RSD1000 may be associated to its voltage-dependent action on I_Na (Fig. 6). Original studies by Woodhull (1973) on a node of Ranvier showed that by increasing extracellular protons (pH_o = 5), peak I_Na was decreased in a voltage-dependent manner to more positive potentials. The model proposed by Woodhull implies that the proton binding site is within the pore and partway across the electric field of the membrane. Woodhull proposed that the proton must move through the field to the get to the site and that the rate constants for binding and unbinding are voltage dependent (Woodhull 1973). Our results at pH 6.4 in the absence of RSD1000 did not reveal a shift of threshold for activation (P > .05) or a reduction in amplitude (P > .05) when compared with pH 7.3 (Fig. 6). The potential for peak I_Na was shifted from −40 to −30 mV (P > .05) when pH was changed from 7.3 to 6.4. In rabbit atrial myocytes, Wendt et al. (1993) reported a positive ∼5 to 10 mV shift in I_Na activation when pH was changed from 7.8 to 6.8. Alternatively, the binding site may not be in the electric field, but rather, the electric field acts on the macromolecule (and on other ions in it) to alter the affinity or availability of the site (Woodhull, 1973). The charged form of RSD1000 at pH 6.4 may perhaps bind to a binding site during the activation process, i.e., through the channel pore, resulting in a voltage-dependent decrease in I_Na (Fig. 9). The voltage-dependent action of RSD1000 arises when the rate-limiting step of the binding process may be the increased energy barrier height of the channel pore, i.e., increased electric field, such that at higher potentials more channels are free and available to conduct. The independence of block at different potentials at pH 7.3 may be due to the neutral from of RSD1000 accessing its binding site via a hydrophobic route. These results may be consistent with those of use-dependent I_Na blocking actions of RSD1000 at different pH_os (Fig. 7), whereby a greater proportion of the charged form at pH 6.4 is present during the activation process. Thus, the pH-dependent action of RSD1000 is voltage-dependent inasmuch as the availability of the binding site is governed by an electric field from changing the membrane potential. Shifts of the I_Na voltage dependence by changes in ionic strength, divalent ion concentration, and pH_o were recognized, therefore, further studies are required to fully elucidate the interaction of RSD1000 and the I_Na channel under different pHs.

The mixed blocking actions of RSD1000 on I_Na and I_TO, as well as potentiated effects on I_Na in acid pH, may be sufficient to explain the selective antiarrhythmic activity of RSD1000 against ischemia-induced arrhythmias. Such a profile would presumably prevent ischemia-induced arrhythmias by virtue of preventing the action potential narrowing due to ischemia and at the same time potentiating the sodium current depressant actions of ischemia. Acting in concert, the actions of RSD1000 would prevent ischemic tissue from participating in re-entry circuits and thereby be antiarrhythmic. The potentiation of action potential modulation in the involved versus uninvolved zone is such that electrical heterogeneity between zones would be unlikely to occur or be reduced. This may explain how RSD1000 prevents ischemia-induced arrhythmias at doses that have no discernible actions on the nonischemic tissue. The fact that the ED₁₀₀ dose for antiarrhythmic protection against ischemia-induced arrhythmias (8 μmol/kg/min) produced some effects on the ECG and on electrically induced arrhythmias does not argue against this, only that RSD1000 has a relative, rather than absolute, selectivity for ischemic tissue.

It would appear, then, that interactions of external H⁺ with protonatable agents possessing pK_as that approximate external pH of ischemic myocardium may serve as one important determinant in the design of pathology-targeted antiarrhythmic agents. This method of drug design appears optimal when the drug pK_a is below physiological pH so as to limit the cationic drug form in nonischemic myocardium but sufficient for ionization in acid pH. Under these conditions, blockade of both sodium and potassium channels may prove more useful than inhibition of a single type of ion channel.