Introduction

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Current antiarrhythmic therapy using drugs is often unsatisfactory, particularly for the severe arrhythmias due to myocardial ischemia-infarction (Roden, 1994; see reviews by Cairns, 1997). There are many reasons for this. One relates to the fact that many of the current drugs provide antiarrhythmic protection by virtue of acting upon on normal (nonpathological) cardiac tissue (Duff et al., 1988; Abraham et al., 1989) to prevent its participation in arrhythmias. Conventional ion channel blocking antiarrhythmics have poor antiarrhythmic efficacy against ischemia/infarction arrhythmias in both clinical (Echt et al., 1991; Waldo et al., 1995) and experimental (Igwemezie et al., 1992; Barrett et al., 1995) settings. This appears to result from the fact that most of such drugs, apart from Class Ib agents, do not select between pathologically disturbed myocardial tissue and normal myocardium. Secondly, because such drugs only act at doses that affect normal cardiac tissue, they are particularly liable to be proarrhythmic (see review by Roden, 1994) because of over expression of their basic action. Furthermore, by virtue of other cardiac and extra cardiac actions they produce toxicities such as cardiac failure and hypertension, plus other unpleasant side effects (Ravid et al., 1989; Schlepper, 1989). Even Class Ib antiarrhythmics, such as lidocaine, lack sufficient ischemia selectivity and thus their therapeutic use is limited by central nervous system toxicity and hypotension (Feldman et al., 1989; Barrett et al., 1995).

Arrhythmias due to myocardial ischemia/infarction depend upon the electrophysiological abnormalities occurring in the pathologically disturbed (ischemic) tissue (Lazzara and Scherlag, 1984; Janse et al., 1986; Kléber, 1986). Thus in the ischemic region, slowing of conduction and decreases in refractoriness (Lazzara and Scherlag, 1984; Janse et al., 1986; Kléber, 1991) create situations whereby reentry circuits between normal and damaged tissues can occur (Lazzara and Scherlag, 1984; Janse et al., 1986; Kléber, 1991). Such reentry circuits can be terminated by either changing electrophysiological behavior in either normal tissue or in the damaged tissue. Changes in electrophysiology in normal tissue are liable to cause arrhythmias and/or depress cardiac functions. Such considerations lead to the suggestion that effective drugs should act selectively on ischemic tissues to first prevent the reduction of refractoriness due to ischemia and then second abolish all electrical activity in the damaged tissues (Walker and Chia, 1989). It may be possible to accomplish this with a drug that selectively acts upon damaged tissues to block both sodium and potassium channels.

The question is how to develop drugs that act selectively on damaged (ischemic) tissue. If one had a drug that blocked ion channels only in its charged form from an external site it would be possible to take advantage of the acid conditions found in the extracellular fluid of ischemic tissue (Abraham et al., 1989; Dennis et al., 1991). Acid conditions could be used to ensure that more of the active species of a drug were available in ischemic as opposed to normal tissue. For example, if such a drug had a pK_a close to the pH of about 6.4 found during ischemia (Owens et al., 1996), then the effective concentration of charged form would be higher in ischemic tissue and selective blockade thereby produced.

RSD1000 [(±)-trans-[2-(4-morpholinyl)cyclohexyl]naphthalene-1-acetate monohydrochloride] is a novel antiarrhythmic agent that blocks sodium and potassium channels and has a pK_a of 6.1 (Fig. 1). We have studied RSD1000 in a variety of rat models to assess both its antiarrhythmic and ion channel-blocking actions. Initial provisional reports have been made of some of the actions of RSD1000 (Yong et al., 1996).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structure of RSD1000; FW: 389.92, (as monohydrochloride salt), 353.54, (as free base); pKa: 6.1.

	Materials and Methods

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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 mM D-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.

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 mM N-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 M. 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 t test 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.

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's t 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 -square test with P < .05. The effects of pH were evaluated by comparing drug changes in acid and normal pH using Student's paired t test.

	Results

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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 and 2). 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 Table 3 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,

(1)

where 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).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Antiarrhythmic dose-response curve for RSD1000 against ischemia-induced arrhythmias. Data is expressed as group mean arrhythmia score of seven experiments. RSD1000 provided dose-related protection against arrhythmias induced by acute myocardial ischemia with an ED50 of 2.5 ± 0.1 µmol/kg/min.

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 3 shows that RSD1000 at infusion levels greater than those in Fig. 2 increased 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.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Actions of RSD1000 on electrical stimulation in vivo. Each point indicates mean of seven experiments. RSD1000 increased threshold currents for induction of VFt (µA) (), iT (µA) (), and ERP (ms) () in a dose-dependent manner. D50%s for VFt, iT, and ERP were estimated to be 15 ± 3.2, 11 ± 1.4, and 7.8 ± 0.9 µmol/kg/min, respectively.

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).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Actions of RSD1000 in isolated rat hearts perfused with normal () or ischemic () buffer (see Materials and Methods). Values are expressed as a percentage change from predrug values (n = 6). In both buffers, RSD1000 increased P-R interval (A) and QRS duration (B) in a concentration-dependent manner. Predrug values for P-R and QRS in ischemic buffer were 83 ± 4 ms and 31 ± 1 ms, respectively, and in normal buffer, 111 ± 2 ms and 34 ± 1 ms, respectively.

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.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Inhibition of INa by RSD1000. A, original INa current traces in the absence (bottom trace) and presence (top trace) of 2 µM RSD1000 at pH 7.3 and 6.4. Holding potential was at 70 mV with depolarizations to 20 mV following an intial prepulse to 140 mV. B, concentration-response plot for effects of RSD1000 on INa peak amplitude. Normalized data are for n = 4 cells studied in bath solution of pH 7.3 () or 6.4 () at the concentrations of RSD1000 shown; C, control.

(2)

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Current-voltage relationship for peak INa under pH 7.3 (A) and 6.4 (B) in control () and in the presence () of 5 µM RSD1000. Peak inward currents were plotted as a function of test potentials elicited from 100 to +30 mV at 10-mV increments at a frequency of 0.5 Hz. Threshold activation of INa for both pH 7.3 and 6.4 was 60 mV with maximum peak INa amplitudes between 40 and 30 mV. At pH 7.3, RSD1000 decreased INa amplitude without changes in threshold and peak potentials. Threshold (50 mV) and peak (20 mV) potentials were both shifted to more positive potentials by RSD1000 in pH 6.4, whereas INa amplitude was decreased (P < .05) to half control maximum.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Use-dependent inhibition of INa. Currents were elicited by a series of 20 depolarizing steps to 20 mV from a holding potential of 100 mV with stimulation frequency of 20 Hz. A, current traces showing actions of 5 µM RSD1000 at pH 7.3 and 6.4. Traces show responses to 1st, 2nd, 5th,10th, and 20th steps of the series. B, plot of normalized INa current as a function of episode number (1-20) at pH 7.3 and 6.4 in the absence (open symbols) and presence (closed symbols) of 5 µM RSD1000.

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 () 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 EC₅₀ was 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  was used as a measure of response (McLarnon and Xu, 1995).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Inhibition of ITO by RSD1000. ITO currents were recorded with a depolarizing step to +60 mV from a holding potential of 70 mV. A, original ITO current traces at pH 7.3 and 6.4 in the absence (top trace) and presence of 2 µM (middle trace) and 30 µM RSD1000 (bottom trace). B, concentration-response curves for decaying time course of ITO by RSD1000, where C denotes control currents. Normalized data are for n = 5 cells, which were studied at both pH 7.3 and 6.4 at concentrations of RSD1000 shown.

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).

View larger version (8K): [in this window] [in a new window]   Fig. 9.   Effects of RSD1000 on ICa. Original traces elicited by depolarizing to +30 mV from a holding potential of 70 mV before and after exposure to 30 µM RSD1000.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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_TO blockade. 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.