INTRODUCTION

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A VARIETY OF THERAPEUTIC strategies have been employed to augment depressed left ventricular (LV) function, which occurs in heart failure and which is particularly intense during periods of decompensation. Catecholamines have been the choice traditionally; however, there are two major factors that limit their effectiveness in heart failure. First, catecholamine desensitization, a characteristic feature of heart failure (2, 13), requires augmentation of the dose continuously to maintain inotropic efficacy. Second, catecholamines increase myocardial oxygen consumption (MO₂) out of proportion to their inotropic action, potentially reducing cardiac efficiency, and may also exert unfavorable effects on membrane stability, resulting in arrhythmias (12, 30). More recently, other pharmacological agents have been used that act distally to cAMP, e.g., on calcium release or myofilament calcium sensitivity. For example, several compounds have been devised that augment the availability of calcium (6, 14-16, 18, 20, 24, 26, 29). One of these, BAY y 5959, is a voltage-dependent Ca²⁺ promoter that exerts a potent effect on LV contractility, with little effect on vasoactivity (1, 25, 31). Importantly, this compound increases myocardial efficiency more than catecholamines for any given amount of cardiac work in normal, conscious dogs (25).

The goal of the current investigation was to determine the effects of the calcium promoter on LV function in the failing heart. Specifically, we wished to determine whether its action was desensitized in heart failure, as occurs with catecholamines. To achieve these goals equi-inotropic doses of BAY y 5959 and dobutamine (Dob) were compared in intact, conscious dogs before heart failure, and the same doses were examined after heart failure was induced by rapid ventricular pacing for 3-4 wk. A secondary goal was to determine whether the new agent, i.e., the calcium promoter, exerted a more beneficial action on cardiac efficiency than Dob, as was observed in conscious dogs without heart failure (25). Accordingly, measurements of arterial and coronary sinus oxygen and coronary blood flow (CBF) were also made and MO₂, cardiac work, and efficiency were calculated. The measurements were compared both in sinus rhythm and with heart rate held constant. Comparisons were made using equi-inotropic doses in the control state before rapid pacing and then in the presence of heart failure and again with equi-inotropic doses. To accomplish the latter, the dose of Dob had to be adjusted in the heart failure state because of catecholamine desensitization.

	MATERIALS AND METHODS

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Surgical preparation. Nine adult mongrel dogs (21-28 kg) of either sex were anesthetized with thiopental (10-20 mg/kg iv) followed by halothane (1.0-1.5 vol%) and ventilated with a Harvard respirator. A left thoracotomy was performed through the fifth intercostal space using sterile technique. Tygon catheters (Norton Elastic and Synthetic Division, Akron, OH) were placed in the descending thoracic aorta and left atrial appendage. A Silastic catheter (Dow Corning, Akron, OH) was implanted in the coronary sinus for the sampling of coronary sinus blood. A solid-state miniature pressure transducer (P6, Konigsberg Instruments, Pasadena, CA) was implanted through the LV apex to measure LV pressure. The piezoelectric ultrasonic dimension crystals were implanted on opposing anterior and posterior endocardial surfaces of the LV to measure LV internal diameter and on opposing endocardial and epicardial surfaces to measure wall thickness. Ultrasonic crystals were also implanted across the LV long axis. Proper alignment of the paired crystals for LV internal diameter, LV long axis, and wall thickness was obtained by positioning the crystals where the greatest amplitude and shortest transit time were observed during surgery. A Transonic flow probe (Transonic Systems, Ithaca, NY) was placed around the left circumflex coronary artery to measure CBF. A Transonic flow probe was also placed around the root of the ascending aorta to measure ascending aortic flow, and a hydraulic occluder (Hazen-Everett, Teaneck, NJ) was also placed around the inferior vena cava. A screw-in pacing lead was attached to the right ventricular free wall, and stainless steel pacing wires were placed on the left atrium. The catheters and lead wires were tunneled subcutaneously to the back of the neck, and the thoracotomy was closed. Each dog was treated with 1 g of cephalothin for 10 days after surgery. The animals used in this study were maintained in accordance with The Guide for Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 83-23, Revised 1985].

Experimental protocols. Experiments were initiated 2-3 wk after recovery from the surgical instrumentation when the dogs were healthy, i.e., body temperature and blood cell count and chemistries were within normal limits. Experiments were conducted in the control state, before pacing, and after heart failure had developed. Dogs were studied in the fully conscious state, while lying quietly in the right lateral recumbent position. All hemodynamic measurements were made in sinus rhythm, and with atrial pacing at 160 beats/min, after a 20- to 30-min stabilization period after the pacemaker was turned off. The aortic and left atrial catheters were connected to strain-gauge manometers (Statham Instruments, Oxnard, CA) for measurements of arterial and left atrial pressures. LV pressure and its first derivative (dP/dt) were measured with a solid-state miniaturized pressure gauge and calibrated in vivo against the measurement of systolic arterial and end-diastolic left atrial pressures. The electrocardiogram was recorded. LV wall thickness and LV diameters were measured with an ultrasonic transit-time dimension gauge (19). The position of all catheters and crystals was confirmed after the animals were killed. In one dog the ultrasonic crystals did not function properly. A Transonic flowmeter was used to measure aortic blood flow and CBF. The flow probe was calibrated in vitro with a timed saline collection in a gravity flow system. In two dogs the aortic blood flow probe did not function properly. A cardiotachometer triggered by the pressure pulse provided instantaneous and continuous records of heart rate. Heart failure was induced by right ventricular pacing at 240 beats/min for 3-4 wk, using a programmable pacemaker (model EV4543, Pace Medical, Waltham, MA) that was worn externally in a vest.

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Data analysis. All hemodynamic data were recorded on a multichannel tape recorder (Honeywell, Denver, CO) and played back on a direct-writing oscillograph (Gould-Brush, Cleveland, OH). LV pressure, LV internal diameter, LV wall thickness, and LV long-axis analog signals were digitized (500 Hz) and analyzed with a computer-based system (Notocord, Croissy, France). LV end diastole was defined as the beginning of positive LV dP/dt. LV end systole was defined as the point of peak negative LV dP/dt. LV fractional shortening was calculated as 100 · [LV end-diastolic internal diameter (EDD)  LV end-systolic internal diameter (ESD)]/EDD (7). The mean velocity of circumferential fiber shortening corrected for heart rate (V_cfc, in s^1/2) was calculated as [(EDD  ESD)/EDD]/[ejection time/R-R interval^1/2 (in seconds)]. Cardiac work was calculated as (MAP  mean left arterial pressure) × cardiac output. MO₂ was calculated from [(arterial O₂ content  coronary sinus O₂ content) × CBF]/100. Cavity volume was calculated with the following formula (22): [(/6)(internal diameter²)(LV long-axis diameter  0.55 wall thickness)]/1,000. The slope of the end-systolic pressure-volume relationship (E_es) was determined from the initial 7-10 beats after occlusion of the inferior vena cava. The point of maximal pressure-to-volume ratio for each cardiac cycle was determined and fitted by a linear regression to determine E_es: end-systolic pressure = E_es[end-systolic volume  intercept of the volume axis (V₀)]. V₀ was estimated by an iterative process until convergence was obtained (11). We calculated mechanical efficiency (Eff) by two different methods, i.e., Eff1 = cardiac work/MO₂ and Eff2 = arterial elastance (E_a) · (end-diastolic volume  V₀)²/[1 + E_es/E_a]²/(MO₂/heart rate) (3, 25). Because the data from two of the dogs could not be used because of malfunction of the aortic blood flow probe, Eff1 was calculated in seven dogs with Dob (10 µg · kg¹ · min¹) and BAY y 5959 (20 µg · kg¹ · min¹) and in five dogs during matched inotropic state. Eff2 was calculated only in the four dogs with an implanted inferior vena cava occluder, because the latter is required to perturb LV pressure. Cardiac work and MO₂ were measured in millimeters of mercury times liter per minute and milliliters of O₂ per minute, respectively, and were converted to joules (1 mmHg · ml = 1.33 × 10⁴ J, and 1 ml O₂ = 20 J).

Statistics. All data are expressed as means ± SE. Because the same animals were used to compare the effects of two agents, a repeated-measures ANOVA procedure of SPSS (SPSS, Chicago, IL) was used to determine statistical significance of the differences between two groups. If the ANOVA demonstrated significant overall differences, individual comparisons between baseline and the response to each drug were made by contrast analysis. A value of P < 0.05 was taken as the minimal level of significance.

	RESULTS

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Comparison of effects of Dob and BAY y 5959 on systemic dynamics and LV function. Before the development of heart failure, Dob (10 µg · kg¹ · min¹) and BAY y 5959 (20 µg · kg¹ · min¹) increased LV systolic pressure, LV dP/dt, and LV fractional shortening similarly (Table 1). Dob increased heart rate, whereas BAY y 5959 decreased heart rate. MAP rose slightly with both drugs but was only significant with BAY y 5959 (Table 2).

View this table: [in this window] [in a new window]   Table 1.   Effects of dobutamine (10 µg · kg1 · min 1) and BAY y 5959 (20 µg · kg1 · min 1) on systemic dynamics before and after heart failure

View this table: [in this window] [in a new window]   Table 2.   Effects of dobutamine (10 µg · kg1 · min 1) and BAY y 5959 (20 µg · kg1 · min 1) on cardiac work, coronary blood flow, myocardial O2 consumption, and mechanical efficiency before and after heart failure

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Dose-dependent effects of BAY y 5959 (left) and dobutamine (Dob; right) on first derivative of left ventricular (LV) pressure (dP/dt, in mmHg/s, n = 9; A) and mean velocity of circumferential fiber shortening (Vcfc, in s1/2, n =8; B) in conscious dogs before and after pacing-induced heart failure. LV dP/dt and Vcfc responses to Dob, but not to BAY y 5959, were desensitized significantly after heart failure. All data are means ± SE. * P < 0.05 for differences in regression lines before and after heart failure.

Comparison of effects of BAY y 5959 and Dob on cardiac output, cardiac work, MO₂, and efficiency. In the control state, in sinus rhythm, Dob increased cardiac output more but stroke volume less than BAY y 5959 because of the positive chronotropic effects of Dob (Table 2). After heart failure, in sinus rhythm, BAY y 5959 increased cardiac output and stroke volume (+115 ± 8%) to a greater degree (P < 0.05) than Dob. With heart rate held constant, with Dob, the cardiac output and stroke volume responses did not differ before versus after heart failure, whereas with BAY y 5959 the cardiac output and stroke volume responses were greater after heart failure compared with before (+80 ± 14 vs. +38 ± 3%, P < 0.05). Furthermore, BAY y 5959 increased cardiac output more than Dob (P < 0.05) after heart failure with heart rate held constant.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effects of Dob (10 µg · kg1 · min1) and BAY y 5959 (20 µg · kg1 · min1) on relation between myocardial oxygen consumption (MO2) and either LV dP/dt (top; n = 9) or Vcfc (bottom; n = 8) in conscious dogs before (A) and after (B) pacing-induced heart failure. Before heart failure, BAY y 5959 and Dob resulted in similar increases in LV dP/dt and Vcfc, but increase in MO2 with BAY y 5959 was significantly smaller than with Dob (P < 0.05). After heart failure, relationship between MO2 and either LV dP/dt or Vcfc remained unchanged for both Dob and BAY y 5959. Increases in LV dP/dt and Vcfc with BAY y 5959 were significantly greater than with Dob (P < 0.05), whereas increase in MO2 with BAY y 5959 was slightly but not significantly smaller than with Dob. All data are means ± SE.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A: pressure-volume loops of baseline in 1 dog in spontaneous rhythm after pacing-induced heart failure. ESPVR, end-systolic pressure-volume relation (dashed line); Ees, slope of ESPVR; Ea, effective arterial elastance; Ved, end-diastolic volume; V0, volume axis intercept of ESPVR line. B: pressure-volume loop at baseline and with BAY y 5959 (20 µg · kg1 · min1; left) and with Dob (10 µg · kg1 · min1; right).

Comparison of effects of BAY y 5959 and Dob with heart rate constant and with inotropic effects matched in heart failure. Because responses to Dob were desensitized after heart failure, it was important to compare the effects of BAY y 5959 and Dob on MO₂ and efficiency under conditions of matched inotropy and with heart rate held constant. The doses selected produced similar increases in LV dP/dt (Dob: +76 ± 7%, BAY y 5959: +83 ± 9%), CBF, and MO₂ in heart failure (Tables 3 and 4). At these doses, increases in %LV shortening, V_cfc, and cardiac output were also similar with BAY y 5959 and Dob. However, MAP increased more (P < 0.05) with BAY y 5959 (+31 ± 7%) than with Dob (+5 ± 4%). This produced a greater increase in cardiac work with BAY y 5959 (+163 ± 11%, P < 0.05) than with Dob (+100 ± 16%). As a consequence, cardiac mechanical efficiency after heart failure, with heart rate held constant and inotropic effect matched, was increased more significantly (P < 0.05) with BAY y 5959 than with Dob. We also calculated mechanical efficiency from pressure-volume relationships with matched inotropy, and with heart rate held constant mechanical efficiency increased significantly (P < 0.05) with BAY y 5959 (+83 ± 33%) but not with Dob (+4 ± 13%).

View this table: [in this window] [in a new window]   Table 3.   Effects of dobutamine (10-15 µg · kg1 · min 1) and BAY y 5959 (10-20 µg · kg1 · min 1) on systemic dynamics during matched inotropic state and with heart rate constant after heart failure

View this table: [in this window] [in a new window]   Table 4.   Effects of dobutamine (10-15 µg · kg1 · min 1) and BAY y 5959 (10-20 µg · kg1 · min 1) on cardiac work, coronary blood flow, myocardial O2 consumption, and mechanical efficiency during matched inotropic state and with heart rate constant after heart failure

	DISCUSSION

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Catecholamine desensitization in heart failure is a critical mechanism, which limits the efficacy of agents such as Dob in the therapy of heart failure. Indeed, in the present study the contractile responses to Dob were reduced by roughly 65% after heart failure. In contrast, the positive inotropic effects of the calcium promoter were not attenuated after heart failure. This lack of desensitization is a major distinguishing feature from the effects of catecholamines and provides an important reason to consider the use of a calcium promoter in the treatment of heart failure. Indeed, desensitization to catecholamines, but not to Ca²⁺, has been noted in human heart failure (4, 5). Other agents that increase the availability or sensitivity to calcium have also been proposed for the treatment of heart failure (14-16, 21, 23, 28). However, most of these agents act by increasing cardiac myofilament sensitivity to Ca²⁺ and also have additional phosphodiesterase III inhibitory activity (17). On the other hand, BAY y 5959 increases Ca²⁺ channel gating by binding dihydropyridine receptors in a voltage-dependent manner, resulting in a reduced rate of Ca²⁺-current activation, increased peak current, and a prolonged tail current decay (1). Nonetheless, it is interesting that Ohte et al. (18) recently observed that the response to pimobendan was also not desensitized in heart failure, consistent with our results for the calcium agonist. Although the two compounds affect calcium handling differently, the end result of preservation of action in heart failure was similar.

Importantly, no prior study has systematically compared the effects of an agent that affects excitation-contraction coupling directly with more traditional sympathomimetic amines before and after heart failure in conscious animals. In the current investigation, BAY y 5959 increased myocardial contractility, as reflected by increases in LV dP/dt, fractional shortening, and V_cfc, in conscious dogs with pacing-induced heart failure. Consequently, BAY y 5959 increased cardiac output by ~50% in spontaneous rhythm (Table 2) and improved cardiac function in the failing heart, despite a modest increase in afterload. The combination of the increase in cardiac output and afterload resulted in a marked increase in cardiac work. All of these factors should produce a significant increase in myocardial oxygen demand. Importantly, BAY y 5959 also improved myocardial O₂ utilization and cardiac mechanical efficiency in the failing heart. This resulted from a proportionately smaller increase in MO₂ compared with increases in cardiac work and contractility. This was most apparent when these actions were compared with those elicited by Dob. The sympathomimetic amine induced greater increases in MO₂ for any given amount of cardiac work or contractility than did the calcium promoter. This is another major reason why a calcium promoter may be useful in the treatment of myocardial depression. Similarly, Ohte et al. (18), using a more indirect index of mechanical efficiency, i.e., stroke work/pressure volume area (PVA), observed that pimobendan increased mechanical efficiency more than amrinone in conscious dogs with heart failure.

The major qualitative difference between the effects of the calcium promoter and Dob before heart failure was the chronotropic response. Even after heart failure Dob either exerted little effect on heart rate or caused it to rise. In contrast, the calcium promoter reduced heart rate significantly in the conscious dogs before and after the development of heart failure. Another recent study from our laboratory (31) demonstrated that the heart rate effect of the calcium promoter was mediated primarily via central and vagal mechanisms. Furthermore, the calcium promoter was able to induce bradycardia in the dogs with heart failure, by preserving the integrity of the baroreflex (31). Regardless of the neural mechanism, it was the bradycardia in the face of increases in cardiac work and contractility that was responsible to a significant extent for the disparity in mechanical efficiency between Dob and BAY y 5959. However, even when heart rate was held constant, the calcium promoter still improved mechanical efficiency to a greater extent than Dob. This was a result of the marked increase in cardiac work with the calcium promoter, without a greater increase in MO₂. In contrast, others have noted that catecholamines result in oxygen wasting (10, 27) that does not appear to be characteristic of the calcium promoter.

Because of the desensitization observed in response to Dob in heart failure, it became important to compare the two drugs at matched increases in myocardial contractility. This was accomplished by comparing higher doses of Dob in the dogs with heart failure. Under these conditions the calcium promoter still utilized oxygen more efficiently, as reflected by the greater increases in mechanical efficiency compared with Dob, supporting the concept that agents that act more directly on excitation-contraction coupling are not oxygen wasting. This is not what was concluded by Hata et al. (9), who examined a different compound but nonetheless one that exerted its major action on excitation-contraction coupling. However, other studies have shown that Ca²⁺ sensitizers decrease (8, 23, 28) or do not change (6, 16) MO₂ in the failing heart. Although Mori et al. (16) and Takaoka et al. (28) demonstrated that the oxygen cost of contractility [i.e., PVA-independent O₂ consumption (O₂)/ slope of pressure-volume relationship (E_max)] was less with the Ca²⁺ sensitizer than with Dob; the mechanical efficiency (i.e., external work/O₂) response to the Ca²⁺ sensitizer was not improved in these patients with LV dysfunction.

In summary, the calcium promoter BAY y 5959 increased cardiac contractility and improved cardiac function. These actions were preserved in heart failure. This preservation of inotropic effect contrasted with the marked desensitization to Dob. In addition, the more favorable effect on myocardial O₂ utilization and cardiac mechanical efficiency in the failing heart also differed from that observed with Dob. These salutary actions may allow this class of drugs to be useful therapeutically in heart failure. Most importantly, the results of this study have more important basic implications, i.e., once calcium is available to the failing heart, its ability to increase inotropy is no longer limited.