INTRODUCTION

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EXERCISE INTOLERANCE is a salient feature of congestive heart failure; the severity of heart failure in patients is classified with respect to exercise intolerance, using the criteria developed by the New York Heart Association (3). Exercise intolerance in heart failure may be based on both an inadequate increase in cardiac output and its distribution into the working skeletal muscle as well as inadequate function and metabolism of skeletal muscle per se (4, 6, 13, 16). Both inadequate perfusion and metabolism of skeletal muscle, in turn, may induce an increase in plasma catecholamine levels (7) and thus contribute to myocardial -adrenoceptor desensitization, arrhythmias, and increased vasomotor tone in other vascular territories (8, 37, 39). In fact, the metaboreflex from skeletal muscle not adequately perfused and metabolically incompetent skeletal muscle may be a predominant source of increased plasma catecholamines (6), rather than arterial and cardiac mechanoreflexes (40). The present study used the established model of pacing-induced heart failure in conscious dogs (5, 10, 11, 14, 17, 20, 22-24, 31, 35). Dogs were subjected to an identical moderate treadmill exercise protocol both in the control state and in heart failure. Cardiac output and its distribution into the working skeletal muscle, including the diaphragm, as well as into the kidney (as a splanchnic reference organ), together with plasma lactate [as an indicator of skeletal muscle anaerobic metabolism (34)] and catecholamines, were measured. To our best knowledge, this is the first study to report such comprehensive analysis of cardiac output distribution, plasma lactate, and catecholamine levels both at rest and during exercise in conscious dogs with pacing-induced heart failure.

	METHODS

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The experimental protocols employed in this study were approved by the bioethical committee of the district of Düsseldorf, Germany, and they conform with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Instrumentation. The experiments were performed in a total of seven adult mongrel dogs (23-36 kg) of either sex. Before instrumentation, all dogs were accustomed to human handling and trained to run on a treadmill.

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Induction of heart failure. After completion of the control studies, a pacemaker (Legend II 8424 and 8426, type VVI; Medtronic) was implanted under local anesthesia (2% lidocaine) in a subcutaneous pouch. Heart failure was induced by rapid LV pacing, as originally reported by Coleman et al. (5). The pacing rate was set to 250 beats/min, and minimal voltage and pulse duration for capture were determined, using a programming unit (model 9710; Medtronic). The presence of heart failure was determined from both clinical signs, such as ascites, pulmonary edema, and cachexia, and hemodynamic parameters, such as the increase in LV end-diastolic pressure (LVEDP) and the decreases in LV maximal pressure (LVP_max), maximum rate of LV pressure rise (LV dP/dt_max), and systolic wall thickening. The mean time (± SD) to induce heart failure was 23 ± 6 days, ranging from 15 to 30 days.

Protocols. Seven to ten days after surgery, when the dogs had fully recovered from surgery, control measurements were performed. Measurements were taken with the dog standing on the treadmill and during exercise. Measurements included the assessment of systemic hemodynamics and regional dimensions, microsphere injection for the assessment of cardiac output and regional blood flow, and arterial blood withdrawal for the analysis of plasma catecholamines and lactate. The dogs were run at a speed of 5 km/h for 10 min. Measurements were performed in a hemodynamic steady state, i.e., between 5 and 10 min into the run.

Measurement of cardiac output and regional blood flow. Cardiac output and regional blood flow were determined with a technique developed in our laboratory (15) using colored microspheres (Dye Trak; Triton Technologies). For each experiment, 8 × 10⁶ to 12 × 10⁶ microspheres suspended in saline solution (containing 0.02% Tween 80) were injected into the left atrium. The number of microspheres was adjusted with respect to the specific absorbance of the respective dye. Simultaneous arterial reference samples were withdrawn from the descending aorta at a constant rate of 5.3 ml/min with a Harvard withdrawal pump (model 901A; Harvard Apparatus, South Natick, MA), starting 30 s before the microsphere injection and continuing for 150 s.

Measurement of catecholamines. For catecholamine analysis, the blood samples were centrifuged immediately after withdrawal, and the plasma was frozen in liquid nitrogen and stored at 70°C. After thawing, 1 ng/ml 3,4-dihydroxybenzylamine was added as an internal standard. The samples were deproteinized (trichloroacetic acid, 0.3 M final concentration), and the catecholamines were adsorbed to purified alumina (1) at pH 8.6 [2.5 M tris(hydroxymethyl)aminomethane · HCl buffer containing 10 mM mercaptoethanol]. The alumina-bound catecholamines were washed three times with 0.8 M acetate buffer, pH 7.5, and once with water. Then the catecholamines were dissolved in 200 ml 1.0 N acetic acid. An aliquot of this solution was used for high-performance liquid chromatography. Separation was achieved on a 3.9 × 150 mm column (Nova-Pak-C18; Millipore, Eschborn, Germany), the mobile phase consisting of 1.0 N acetic acid, 22 mM 1-octanesulfonic acid sodium salt monohydrate, 0.2 mM EDTA-Na₂, with a flow of 1.3 ml/min (pump: Waters M510, Millipore; pulse dampener: LP-21, Scientific Systems, State College, PA). A glassy carbon electrode set to 650 mV against Ag-AgCl was used for electrochemical detection (model 400 EC Detector; EG & G Princeton Applied Research, Princeton, NJ).

Measurement of lactate. Lactate was determined from KF-EDTA-stabilized blood samples, using the lactate dehydrogenase reaction combined with transamination of the formed pyruvate for quantitative NADH formation (18). NADH was measured photometrically.

Data analysis and statistics. LVP, LV rate of pressure rise (LV dP/dt), and regional wall thickness were simultaneously recorded on an eight-channel recorder (model RS800; Gould, Cleveland, OH) and a personal computer. Hemodynamic data were digitized at a rate of 200 Hz and directly stored on hard disk with the use of Cordat II software (28). Systemic hemodynamic and regional wall function data of 15 sequential beats were averaged. End diastole was defined as the time point at which LV dP/dt started its rapid upstroke after crossing the zero line. Regional end systole was defined as the point of maximal wall excursion within 20 ms before peak negative dP/dt. The measured parameters were heart rate, LVP_max and LVEDP, LV dP/dt_max, end-diastolic wall thickness, and systolic wall thickening, presented as the percent change from the end-diastolic thickness. End-diastolic wall thickness was normalized to 10 mm under control conditions (19).

	RESULTS

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Baseline hemodynamics. After 3-4 wk of rapid ventricular pacing, dogs developed heart failure, characterized by clinical signs, such as ascites, pulmonary edema, and cachexia (body weight decreased from 25.6 ± 3.4 to 22.4 ± 2.3 kg), and hemodynamic parameters, such as an increase in LVEDP and decreases in LVP_max, dP/dt_max, and systolic wall thickening.

Hemodynamic responses to exercise. In the control state, during moderate exercise, there were significant increases in heart rate, LVP_max, dP/dt_max, and cardiac output (Fig. 1; Table 1). In heart failure, starting from reduced levels at rest, there were still significant increases in LVP_max and dP/dt_max. Heart rate was not different at rest and increased to a similar extent as during control exercise. LVEDP at rest was significantly elevated and increased further during exercise. Cardiac output at rest was markedly reduced in heart failure, and there was no significant increase during exercise (Table 1). In contrast to the control state, all dogs in heart failure had obvious signs of discomfort during exercise.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Representative original recordings of hemodynamic responses to treadmill exercise at 5 km/h. LVP, left ventricular pressure; LV dP/dt, rate of left ventricular pressure rise (first derivative of LVP); WT, wall thickness.

Myocardial, skeletal muscle, and renal blood flow. In the control state, blood flow to the myocardium, the working skeletal muscle, including the diaphragm, and the kidney increased during exercise (Table 2). The increase in blood flow to the biceps femoris was most pronounced, whereas the increase in renal blood flow was only modest. In heart failure, blood flow at rest was reduced in all organs studied, particularly in the skeletal muscle. Also, the increases in myocardial and skeletal muscle blood flow during exercise were blunted. In heart failure, renal blood flow was no longer increased.

Plasma catecholamines and lactate. In the control state, plasma catecholamines and lactate were increased slightly during exercise (Table 3). In heart failure, plasma catecholamines were increased at rest and increased more markedly during exercise. Also, lactate increased markedly during exercise in heart failure.

	DISCUSSION

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The present study utilized an established experimental model of heart failure, i.e., chronic rapid ventricular pacing in conscious dogs (5, 10, 14, 17, 20, 22-24, 31, 35). This model mimics not only the clinical signs (ascites, pulmonary edema, cachexia) seen in patients with heart failure but also the associated hemodynamic and neurohumoral alterations (increases in LV end-diastolic pressure and plasma catecholamines, decreases in LV maximal pressure, dP/dt_max, and cardiac output).

The exact mechanisms underlying the development of heart failure with chronic rapid ventricular pacing are currently unclear (27) but may be related to abnormalities of intracellular calcium kinetics (20) and myocyte loss (11). To that extent, it is currently unclear whether pacing-induced heart failure shares the same pathophysiology as heart failure in humans. Important for the present study, however, is that exercise intolerance, which is a salient feature of heart failure in humans, is also evident in the pacing-induced heart failure model. The fairly mild severity of heart failure in the present study was similar to that previously reported by others, as judged by the impairment in baseline hemodynamics (2, 5, 11, 14, 20, 22-24, 31, 35) as well as by the increase in resting plasma catecholamines (2, 9, 21). The reduction in cardiac output at rest by an average of 44% in the present study was even more severe than in several prior studies (9, 14, 35), comparable only with that reported by Moe et al. (17).

From a comparison of the baseline data in the control state of the present study with those previously reported (9, 24), it appears from the catecholamine levels that dogs were under some adrenergic activation already at rest, possibly in anticipation of the run. This adrenergic activation was also associated with somewhat higher skeletal muscle blood flow levels compared with the study of Shen et al. (26). In contrast, the intensity of exercise in the present study was fairly mild, as reflected by the only modest increases in plasma catecholamines and lactate as well as the hemodynamic responses under control conditions. More significant increases in plasma catecholamines and lactate were seen only during such mild exercise after the development of heart failure. Such mild exercise intensity was chosen so that dogs could perform an identical exercise protocol in the control state and in heart failure.

Flow measurements in the present study were based on the microspheres technique. We did not attempt to address a potential loss of microspheres over time in the present study. However, from previous studies using the model of pacing-induced heart failure in dogs and swine, no loss of microspheres was reported (25, 29, 30). Also, we did not perform morphological studies. However, prior studies using this model have not reported an increase either in LV muscle mass or in capillary density and cross-sectional area (25, 29).

In heart failure, blood flow to the myocardium at rest was somewhat less reduced (LV 30% on average; right ventricular 20% on average) than cardiac output (44% on average). Also, blood flow to the renal cortex was better maintained than cardiac output (28% on average). In contrast, blood flow to the skeletal muscle at rest, including the diaphragm, was much more markedly reduced (triceps 62%; biceps femoris 62%; diaphragm 68%). During exercise, the decrease in cardiac output by 62% on average was even more pronounced compared with rest (44%), and the same was true for LV myocardial blood flow (44% vs. 30% at rest). In contrast, the decreases in skeletal muscle blood flow during exercise were much less pronounced than those in cardiac output (triceps 25%; biceps femoris 45%; diaphragm 33%). Apparently, in heart failure there is a preferential distribution of cardiac output into the working skeletal muscle during exercise, as also evidenced by the concomitant albeit not significant decrease in renal blood flow seen in the present study. No increase in renal blood flow during exercise in the control state and a marked decrease in renal blood flow in exercising dogs with heart failure caused by tricuspid avulsion and pulmonary stenosis were previously reported (8). However, that particular study differed from the present one with respect to the heart failure model (tricuspid avulsion and pulmonary stenosis vs. pacing), exercise severity (heart rate of 281 ± 25 vs. 166 ± 19 beats/min), and method of flow measurement (Doppler flow probe vs. microspheres). Despite these differences, both studies indicate a reversal of the renal blood flow response to exercise with heart failure. Even with preferential perfusion (relative to cardiac output) of the working skeletal muscle, there was a significant increase in plasma lactate, supporting the notion that the skeletal muscle in heart failure is characterized not only by reduced perfusion (in absolute terms) but also by primary metabolic abnormalities (13, 16). However, we did not determine the exact source of increased plasma lactate (e.g., liver vs. skeletal muscle).

The coronary circulation in this heart failure model is under defective endothelial control (33). Reduced myocardial blood flow at rest and, in addition, in adenosine- or atrial pacing-recruitable coronary reserve has been reported previously in conscious dogs (25) and pigs (29) with pacing-induced heart failure. The impairment in coronary reserve in dogs was selective to the subendocardium (25). In the present study, however, a preferential impairment of subendocardial blood flow was not observed either at rest or during exercise, although extravascular compression, as reflected by LV end-diastolic pressure, was increased. Obviously, at this mild exercise level, an adequate coronary vasomotor response to increased cardiac work was still possible, indicating no enhanced vulnerability of the failing myocardium to ischemia.

Unfortunately, arterial pressure was not measured in the present study, since the arterial line was used for the reference withdrawal during the microsphere measurement of blood flow. Therefore, resistance values cannot be calculated and pressure-dependent changes in blood flow cannot be distinguished from active vasomotion. In particular, the attenuated increases in flow to myocardium and skeletal muscle during exercise and, even more so, the lack of flow increase to the kidney may, in part, be caused by an attenuated increase in arterial pressure, as has been reported in patients with heart failure during exercise (36).

A limitation of the present study is the lack of mechanistic information on the causal relation among hemodynamics, blood flow distribution, plasma catecholamines, and lactate. However, this is the first study to provide integrative information on these parameters during exercise in the model of pacing-induced heart failure. Inadequate nutritive perfusion of skeletal muscle during exercise also has been reported in patients with heart failure (32, 36, 38) and has been related to impaired endothelial function (12). Also, increased lactate production during exercise has been reported in patients with heart failure (32, 36). With respect to the symptom of exercise intolerance and the observed phenotype of hemodynamic, blood flow, plasma catecholamine, and lactate responses to treadmill exercise, the pacing-induced heart failure model in conscious dogs may therefore be useful to analyze the mechanisms of exercise intolerance in future studies.