Introduction

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A variety of therapeutic strategies have been applied to augment the depressed left ventricular function of heart failure, particularly during periods of acute decompensation (Packer, 1988; Rahimtoola, 1989; Shipley and Hess, 1995). Current clinically available inotropic agents that act via elevation of cyclic AMP, such as dobutamine and phosphodiesterase inhibitors; are associated with tachycardia, significant changes in preload or afterload or increases in myocardial O₂ consumption (MVO₂), and decreases in myocardial mechanical efficiency (Katz, 1986; Simaan et al., 1988). Recently, there has been interest in agents that either increase myofibrillar Ca²⁺ availability or directly sensitize myocardial cells to Ca²⁺ without the participation of cAMP (Ruegg, 1986; Doggrell et al., 1994; Mathew and Katz, 1998). One strategy has been the development of Na⁺ channel enhancers that prolong the open state of Na⁺ channels, increasing net Na⁺ influx and the activity of reverse mode Na⁺/Ca²⁺ exchange (for review, see Steinberg et al., 1998). This leads to a decrease in net Ca²⁺ efflux and increases in intracellular free Ca²⁺ (Romey et al., 1987; Scholtysik, 1989). Sodium channel enhancers have been shown to elicit positive inotropic effects in isolated cardiac tissues from experimental animals and from human papillary muscles obtained from normal as well as failing hearts (Flesch et al., 1996; Schwinger et al., 1996; Müller-Ehmsen et al., 1997). These compounds also improve left ventricular contractile function in anesthetized dogs with either normal (Baumgart et al., 1994) or failing hearts (Taniumra et al., 2000).

Recently, we found that Na⁺ channel enhancers increased myocardial contractility in conscious normal dogs without increasing heart rate, consequently resulting in an enhancement of cardiac mechanical efficiency for a given cardiac workload (Shen et al., 2001; Gill et al., 2002), suggesting a potential benefit in heart failure. The primary goal of the present investigation was to determine the effects of the Na⁺ channel enhancer, LY341311, on LV function in conscious dogs with pacing-induced heart failure. Second, we wanted to determine whether the lack of heart rate effect previously seen in normal dogs extends to dogs with heart failure and whether the negative chronotropic responses to LY341311 could mediate potential beneficial effects on myocardial O₂ consumption and cardiac efficiency in the failing heart. Myocardial and hemodynamic effects of LY341311 were compared with dobutamine, a standard positive inotropic agent that acts via the -receptor to increase intracellular cyclic AMP (Tuttle and Mills, 1975).

	Materials and Methods

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Animal and Surgical Preparation. Male adult mongrel dogs (20-30 kg) were used for this study. Dogs were anesthetized with isoflurane in oxygen and ventilated with a respirator after induction with acepromazine (0.03 mg/kg i.m.) and propofol (5.5 mg/kg i.v.). A left thoracotomy was performed through the fifth intercostal space under sterile technique. Tygon catheters were implanted in the descending thoracic aorta, left atrial appendage, and left ventricles for measuring pressures. A Silastic catheter was implanted in the coronary sinus for the sampling of coronary venous blood. A solid-state miniature pressure transducer (model P6; Konigsberg, Pasadena, CA) was placed into the left ventricular chamber via an apical stab incision for recording left ventricular pressure (LVP). A Transonic flow probe (Transonic Systems, Inc., Ithaca, NY) was implanted around the left circumflex coronary artery for measuring coronary blood flow (CBF). A pair of piezoelectric ultrasonic crystals was placed on opposing anterior and posterior endocardial surfaces of the left ventricle for measuring LV i.d. A screw-in pacing lead was attached to the right ventricular free wall, and stainless steel pacing wires were placed on the left atrium. All instruments were secured with sutures. The catheters and lead wires from the instruments were externalized, the thoracotomy was closed in layers, and the intrapleural space was evacuated. Antibiotics were administrated postoperatively [with cephalexin (500 mg)] for 7 days following surgery. Control experiments were initiated 2 to 3 weeks after surgery when the dogs were healthy, i.e., body temperature, blood cell count, and chemistries were within normal limits.

Canine Model of Chronic Heart Failure. After the initial control study, heart failure was induced by chronic rapid right ventricular pacing at a rate of 240 bpm for 3 to 4 weeks, using a programmable pacemaker (model EV4543; Pace Medical, Waltham, MA) that was worn externally in a vest (Shen et al., 1996; Asai et al., 1998; Shen et al., 1999).

Measurements and Data Analysis. All measurements were made in dogs in the full conscious state, while lying quietly in the right lateral recumbent position. Studies in heart failure were conducted in experimental dogs after a 30-min stabilization period after the pacemaker was turned off. Hemodynamic signals were collected on-line and analyzed on a beat-to-beat basis using a digital data acquisition system (PONEMAH; Gould Instrument System, Inc., Cleveland, OH). The sampling rate was 250 Hz for arterial pressure, left atrial pressure (LAP), and CBF, and 500 Hz for LVP and LV i.d.

Materials. LY341311 (Fig. 1), [(S)-4-[3-[[1-(diphenylmethyl)-3-azetidinyl]oxy]-2-hydroxypropoxy]-1H-indole-2-carbonitrile monohydrate)], is also referred to elsewhere as BDF9196 or as LY366634 (the anhydrous free base). LY341311 was dissolved (5 mg/ml) in a stock solution containing 1% (v/v) glacial acetic acid, 2% (v/v) absolute ethanol, 2.5% (w/v) L-ascorbic acid, and distilled water to volume. The stock was then diluted in 5% dextrose solution (Abbott Laboratories, Chicago, IL). Dobutamine (Bedford Laboratories, Bedford, OH), hexamethonium (Sigma-Aldrich, St. Louis, MO), and atropine methyl bromide (Sigma) were diluted in 0.9% NaCl saline.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   The structure of LY341311, (S)-4-[3-[[1-(diphenylmethyl)-3-azetidinyl]oxy]-2-hydroxypropoxy]-1H-indole-2-carbonitrile monohydrate, also known and previously referred to as BDF9196 or LY366634 (the free base).

Experimental Protocols. LY341311 was administered as graded intravenous infusions of 10, 25, 50, and 100 µg/kg/min for 15 min at each dose level. A vehicle control for LY341311 was also performed in five dogs. Dobutamine was administrated as a 5-min graded intravenous infusion of 2, 5, 10, and 15 µg/kg/min. The dose and infusion time for each drug were based on preliminary studies to confirm that steady-state levels of responses were achieved with this dosing protocol. In five dogs the responses to LY341311 (100 µg/kg/min) and dobutamine (10-15 µg/kg/min) were also examined during left atrial pacing (150 bpm for 5 min) under conditions of matched inotropy and cardiac performance. The cardiac and hemodynamic responses to LY341311 were also examined following pretreatment with ganglionic blockade, by either the combination of hexamethonium (30 mg/kg i.v. for 20 min) and atropine methyl bromide (0.1 mg/kg i.v.) (n = 5) or hexamethonium alone (30 mg/kg i.v. for 20 min) (n = 3). Hemodynamic and cardiac functional data were recorded throughout the experiments, and blood samples for the measurement of MVO₂ were taken at baseline and after each dose of LY341311 or dobutamine when a steady state was reached in the presence or absence of pacing.

Statistical Analysis. For hemodynamic, cardiac function, and MVO₂ data, values are expressed as mean ± S.E., and a one-way factorial analysis of variance was used to determine the overall significance of differences. If the analysis of variance demonstrated significant overall differences, individual comparisons between baseline and the response to each drug were made by contrast analysis. All changes were considered significant when P < 0.05 using a two-tailed t distribution.

	Results

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Hemodynamics and Cardiac Function in Conscious Dogs with Heart Failure. Hemodynamics and cardiac function were evaluated in a conscious state before and after the development of heart failure (Table 1). Rapid right ventricular pacing at 240 bpm for 3 to 4 weeks resulted in significant decreases in LV systolic and diastolic function, including decreases in LV dP/dt_max, LV dP/dt/40, LV %FS, LV dP/dt_min, and increased LV relaxation . An increase in LAP, LVEDD, LVEDP, and heart rate, and a decrease in MAP accompanied the decrease in cardiac contractility and relaxation (Table 1). Exertional dyspnea and ascites also were observed. All hemodynamic cardiac functional data and clinical signs indicated the development of severe congestive heart failure, consistent with previous studies (Shen et al., 1996, 1999).

Effect of LY341311 on Cardiac Systolic and Diastolic Function. In conscious dogs with pacing-induced heart failure, LY341311 (10 to 100 µg/kg/min) caused dose-dependent increases in cardiac contractile performance, represented by increased LV dP/dt_max and LV %FS (Fig. 2), with no change in the vehicle group. Compared with baseline, LY341311 (100 µg/kg/min) increased LVSP by 15 ± 2 mm Hg, LV dP/dt_max by 746 ± 77 mm Hg/s, dP/dt/40 by 536 ± 80 s¹, and LV ejection phase %FS by 6.5 ± 1.0% and decreased LVESD by 2.5 ± 0.4 mm (all P < 0.05), without significant change in mLAP (0.8 ± 1.1 mm Hg), LVEDP (0.6 ± 1.2 mm Hg), and LVEDD (0.3 ± 0.2 mm). Consequently, LY341311 increased LV stroke work by 47 ± 7 × 10³ dyne/cm and LV minute work by 1.6 ± 0.5 × 10⁶ dyne/cm/min (Table 2). These data indicate a positive cardiac inotropic effect of LY341311 in the absence of significant changes in preload as assessed by LV end-diastolic pressure and dimension (Table 2), in conscious dogs with pacing-induced heart failure. LY341311 also caused dose-dependent increases in cardiac relaxation, as indicated by increased LV dP/dt_min and decreased LV relaxation  (Fig. 3). Compared with baseline, LY341311 (100 µg/kg/min) increased LV dP/dt_min by 479 ± 45 mm Hg/s and LV relaxation  by 6.0 ± 1.3 s¹ (Table 2).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Dose-dependent effects of LY341311 (left, ) and dobutamine (right, ) on LV dP/dtmax (n = 10) and fractional shortening (n = 8) in conscious dogs with heart failure. Similar to dobutamine, LY341311 increased LV dP/dtmax and fractional shortening. There was no response to the vehicle for LY341311 (). All data are mean ± S.E. *, P < 0.05 versus baseline.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Dose-dependent effects of LY341311 (left, ) and dobutamine (right, ) on LV dP/dtmin and LV relaxation  in conscious dogs with heart failure (n = 10). Both LY341311 and dobutamine increased LV dP/dtmin and reduced LV relaxation . There was no response to the vehicle for LY341311 (). All data are mean ± S.E. *, P < 0.05 versus baseline.

The Autonomically Mediated Negative Chronotropic Effect of LY341311. A major difference between LY341311 and dobutamine in conscious dogs with heart failure related to their chronotropic response (Fig. 4). Heart rate was reduced by 41 ± 7 bpm (35 ± 5%) with LY341311 at the dose of 100 µg/kg/min, whereas dobutamine did not change heart rate at the dose of 10 µg/kg/min (4 ± 5 bpm) and increased heart rate by 17 ± 5 bpm (15 ± 5%) at the dose of 15 µg/kg/min (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of LY341311 (left, ) and dobutamine (right, ) on heart rate (n = 10) and MVO2 (n = 8) in conscious dogs with heart failure. Dobutamine dose dependently increased both heart rate and MVO2. In contrast, LY341311 caused a decrease in heart rate and no significant change in MVO2. There was no response to the vehicle for LY341311 (). All data are mean ± S.E. *, P < 0.05 versus baseline.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of LY341311 on heart rate, LV dP/dtmax, dP/dtmin, and LV relaxation  following pretreatment with ganglionic blockade (GB) [hexamethonium (30 mg/kg i.v. for 20 min)] alone or with atropine methyl bromide (0.1 mg/kg i.v.)] in conscious dogs with heart failure (n = 8). After autonomic block, the negative chronotropic response to LY341311 was abolished, but the increase in LV dP/dtmax, dP/dtmin, and LV relaxation  to LY341311 was maintained. All data are mean ± S.E. *, P < 0.05 versus baseline.

Effects of LY341311 on Coronary Blood Flow, Myocardial O₂ Consumption, and Cardiac Mechanical Efficiency. LY341311 did not change myocardial O₂ consumption, despite its positive cardiac inotropic effect (Fig. 4). LY341311 at the dose of 100 µg/kg/min, which caused significant increases in LV dP/dt and LV %FS, only increased mCBF and MVO₂ by 2.7 ± 2.0 ml/min and 0.1 ± 0.3 ml/min, respectively (Table 4). This was accompanied by a decrease in myocardial O₂ extraction of 10 ± 3% (P < 0.05), but without significant changes in blood hematocrit and arterial blood total O₂ content (Table 4). In contrast, dobutamine caused a significant increase in myocardial O₂ consumption with its positive cardiac inotropic response (Fig. 4). Dobutamine (10 µg/kg/min) produced an inotropic effect similar to that of LY341311 (100 µg/kg/min), and increased mCBF by 10 ± 2 ml/min and MVO₂ by 1.1 ± 0.2 ml/min (both P < 0.05 versus LY341311). Dobutamine also increased blood hematocrit by 5 ± 2% (P < 0.05) and arterial blood total O₂ content by 7 ± 2% (P < 0.05), without significant change in myocardial O₂ extraction (Table 4). Thus, under similar inotropic states, mCBF and MVO₂ were 11% and 31% higher with dobutamine compared with LY341311 in the conscious dog with heart failure.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of LY341311 () and dobutamine () on the relation between MVO2 and either LV dP/dt or LV fractional shortening in conscious dogs with heart failure (n = 8). LY341311 and dobutamine resulted in similar increases in LV dP/dt and fractional shortening, but MVO2 increased only with dobutamine and not LY341311. All data are mean ± S.E. The slopes relating MVO2 to either LV dP/dt or LV fractional shortening were significantly different comparing dobutamine with LY341311 (P < 0.05).

Comparison of Effects of LY341311 and Dobutamine on MVO₂ with Constant Heart Rate and Matched Inotropy. To understand the potential mechanisms that may be responsible for the lower MVO₂ and greater efficiency of LY341311 compared with dobutamine, the effects of both compounds on MVO₂ were compared under conditions of matched inotropy at constant heart rate. Matched inotropy was produced at an LY341311 dose of 100 µg/kg/min and a dobutamine dose of 10 to 15 µg/kg/min, and constant heart rate was achieved by left atrial pacing at 150 bpm. At these doses, increases from baseline in LV dP/dt_max and LV %FS with LY341311 or dobutamine were similar (Fig. 7). From similar baselines, MVO₂ was elevated significantly by dobutamine during sinus rhythm and did not increase further after pacing, whereas MVO₂ was unchanged by LY341311 and increased significantly during pacing (Fig. 7). The MVO₂ with LY341311 during pacing reached the same level as dobutamine.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effects of LY341311 () and dobutamine () on MVO2 were compared in conscious dogs with heart failure (n = 5) under conditions of matched inotropy at constant heart rate (150 bpm). LY341311 (100 µg/kg/min) and dobutamine (10-15 µg/kg/min) produced a matched inotropy, represented by LV dP/dt (left) and fractional shortening (center). In contrast to dobutamine, MVO2 with LY341311 was only slightly elevated in sinus rhythm but was increased during pacing to the level equivalent to dobutamine. All data are mean ± S.E. BL is pretreatment baseline. *, P < 0.05 versus pretreatment baseline; #, P < 0.05 versus dobutamine.

Effects of LY341311 on Electrocardiographic Parameters. The influence of LY341311 on electrocardiographic parameters in dogs with heart failure was evaluated in the conscious state. The dose-dependent decrease in heart rate resulted as expected in increases of the P-R and the Q-T intervals; however there was no change in the QRS duration or the Q-Tc interval (Table 5).

	Discussion

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Synthetic sodium channel enhancers prolong the open state of the Na⁺ channel, probably by inhibiting Na⁺ channel inactivation, thereby increasing net Na⁺ influx. The increased intracellular Na⁺ concentration, in turn, activates reverse-mode Na⁺/Ca²⁺ exchange to augment intracellular Ca²⁺, leading to a positive inotropic effect (Scholtysik, 1989). Numerous studies in vitro have demonstrated that Na⁺ channel enhancers, including LY341311, elicit positive inotropic effects on isolated cardiac papillary and ventricular muscles from rats, from guinea pigs, and from normal and failed human hearts (Brasch and Iven, 1991; Ravens et al., 1991; Flesch et al., 1996; Nand and Doggrell, 1999). Studies in vivo have also shown that Na⁺ channel enhancers increase cardiac contractile performance in anesthetized dogs with acute ischemia (Baumgart et al., 1994) and in the chronic microembolization-induced failing heart model (Taniumra et al., 2000). However, no prior study has systematically investigated the effect of a Na⁺ channel enhancer on hemodynamic and cardiac function in the conscious animal with heart failure. In the current study, the Na⁺ channel enhancer, LY341311, increased myocardial contractility and contractile performance, as reflected by increasing LV dP/dt_max, LV dP/dt/40, and LV %FS without a significant change in preload. Consequently, cardiac stroke work increased by 111 ± 18% and cardiac minute work increased by 34 ± 10%, as estimated from LV pressure-dimension loops in spontaneous rhythm. Our results indicate a significant positive cardiac inotropic effect of LY341311 in the failing heart, thereby improving cardiac pump function in conscious dogs with heart failure. These results were consistent with previous in vitro and in vivo studies.

The major qualitative difference between the effects of LY341311 and dobutamine in the conscious dog with heart failure was the cardiac chronotropic response. Dobutamine exerted either little effect on heart rate or increased heart rate at the highest dose, whereas LY341311 resulted in a dose-dependent decrease in heart rate, which had not been previously reported. If the negative cardiac chronotropic effect of LY341311 were neurally mediated, the responses to LY341311 would be expected to diminish in the presence of autonomic block. Indeed, the cardiac negative chronotropic effect was completely eliminated in the presence of ganglionic blockade, whereas the cardiac inotropic response to LY341311 was maintained. Thus, the negative chronotropic response to LY341311 is autonomically mediated, but the increase in cardiac contractility was a direct effect of LY341311 on the myocardium, whereas the direct inotropic action likely related to the inhibition of Na⁺ channel inactivation. The mechanism responsible for the negative cardiac chronotropic effect of the Na⁺ channel enhancer is not clear. One possibility is an improvement in the sensitivity of the baroreflex arc by the increased receptor sensitivity due to local alterations in the Na⁺ content of the vessel wall (Kunze et al., 1978). It has also been reported that inotropic agents that increase cellular calcium may act centrally to increase vagal tone in dogs with heart failure (Uechi et al., 1998). Interestingly, the negative cardiac chronotropic response to LY341311 was not found in normal dogs, either under anesthetic (Baumgart et al., 1994) or fully conscious (Gill et al., 2002). It is well known that heart failure is associated with derangements of autonomic control of the cardiovascular system, manifested as reduced parasympathetic activity and elevated sympathetic activity (Kinugawa and Dibner-Dunlap, 1995; Wang et al., 1999). Thus, our data suggest that the Na⁺ channel enhancer might specifically facilitate a rebalancing of autonomic tone in heart failure.

Since heart rate is one of the critical factors contributing to MVO₂, and LY341311 and dobutamine had opposite chronotropic responses, we hypothesized that the lower MVO₂ with LY341311 was mainly due to its negative cardiac chronotropic effect. Indeed, the difference in MVO₂ between LY341311 and dobutamine at similar myocardial contractility and cardiac work was abolished when heart rate was held constant at 150 bpm by atrial pacing. Thus, the negative chronotropic effect of LY341311 appears to be a major mechanism contributing to the lower myocardial O₂ cost during its inotropic effect in conscious dogs with heart failure. In fact, the reduction of heart rate has been suggested as one of the major beneficial mechanisms for -blockade to improve cardiac function in heart failure (Packer et al., 1996; Nagatsu et al., 2000). Although the elevation of heart rate in heart failure represents a compensatory mechanism via increased sympathetic activity, tachycardia itself can cause further deleterious effects on the failing heart (Shinbane et al., 1997; Nikolaidis et al., 2001).

One of the major findings from the current study related to an improved myocardial O₂ utilization and cardiac mechanical efficiency in the failing heart by LY341311. This resulted from a proportionately smaller increase in MVO₂ compared with increases in myocardial contractility and cardiac work. Dobutamine caused a greater increase in MVO₂ for any given level of myocardial contractility and cardiac work than did the Na⁺ channel enhancer. Since impaired energy metabolism is directly associated with the development of heart failure (Shen et al., 1999), preventing further deterioration of myocardial energetics while attempting to correct cardiac decompensation in the failing heart is an important goal. Our results demonstrated that LY341311 improved cardiac mechanical efficiency and mitigated the expected increase of MVO₂ due to enhanced myocardial contractile performance, thereby providing a favorable cardiac energetic benefit to the failing heart.

Although Na⁺ channel enhancers indirectly increase intracellular Ca²⁺ availability (Romey et al., 1987; Scholtysik, 1989), this intracellular Ca²⁺ gain might be expected to slow sacroplasmic reticulum Ca²⁺ uptake and have adverse effect on cardiac diastolic function. Interestingly, this did not appear to be the case in our study. On the contrary, LY341311 improved myocardial diastolic relaxation, as reflected by increases in LV dP/dt_min by 31 ± 3% and reductions in LV  by 19 ± 4% during sinus rhythm. Furthermore, the improvement of myocardial diastolic relaxation with LY341311 became more pronounced after the negative chronotropic response of LY341311 was attenuated with ganglionic blockade (Table 3), since relaxation indices are known to be slowed by bradycardia (Weiss et al., 1976; Asanoi et al., 1996). Thus, LY341311 appears to exert not only positive inotropic but also positive lusitropic effects in conscious dogs with heart failure. Other investigators have shown that Na⁺ channel enhancers augmented contractile performance without increasing diastolic tension in isolated papillary muscle from normal as well as failing human hearts (Flesch et al., 1996). These investigators suggested that a reduction of the Na⁺ gradient might cause increases in both Ca²⁺ influx and efflux; the latter could be secondary to an enhanced release of Ca²⁺ from sarcoplasmic reticulum (Bers, 1987). This could also be a contributing factor to the positive lusitropic effect of the Na⁺ channel enhancer seen in our study.

Cardiac glycosides also increase intracellular Na⁺ concentration through inhibition of the Na⁺/K⁺-ATPase, to produce positive cardiac inotropic effects. However, cardiac glycosides can induce or aggravate arrhythmia and thus possess a very narrow therapeutic index (Kelly and Smith, 1993). In this regard, it was reported that the Na⁺ channel enhancers DPI 201-106 and BDF 9148 displayed proarrhythmic potential in anesthetized dogs with myocardial infarction (Stump et al., 2001). LY341311 caused dose-dependent increases in the P-R and QT intervals, whereas heart rate was significantly reduced from 115 ± 5 to 76 ± 5 bpm. There was no prolongation of the QRS duration or Q-Tc interval with any dose of LY341311. The electrocardiographic interval data from the conscious dogs with heart failure are consistent with the bradycardia, and we saw no evidence for an arrhythmogenicity. Nevertheless, this study was not specifically designed to address any arrhythmogenic potential of LY341311, and further investigation is clearly warranted.

In summary, the novel Na⁺ channel enhancer LY341311 increased cardiac contractility and relaxation, and improved cardiac function in conscious dogs with pacing-induced heart failure. In contrast to dobutamine, LY341311 caused an autonomically mediated negative chronotropic response that favorably modulates the balance of sympathetic and parasympathetic control of heart rate. Consequently, the negative chronotropic response appears to be a major mechanism contributing to the favorable metabolic effect in the failing heart while maintaining the cardiac inotropic effect needed for the correction of depressed ventricular cardiac function.