Inhalation of the Phosphodiesterase-3 Inhibitor Milrinone Attenuates Pulmonary Hypertension in a Rat Model of Congestive Heart Failure

All experiments were performed in male Sprague-Dawley rats. Animals received care in accordance with the Guide for the Care and Use of Laboratory Animals .13The study was approved by the local animal care and use committee of the local government authorities (Tierversuchskommission, LAGetSi Berlin, Germany).

In juvenile rats of 100 ± 8 g body weight (bw), CHF was induced by supracoronary aortic banding as previously described.14In brief, rats were anesthetized by intraperitoneal injection of ketamine (87 mg/kg bw; Pharmacia GmbH, Erlangen, Germany) and xylazine (13 mg/kg bw; Bayer, Leverkusen, Germany). Rats were placed in the supine position, the chest wall was shaved, and a left thoracotomy was performed in the third intercostal space during ventilation with 100% O². The ascending aorta was freed from connective tissue and partially occluded by implantation of a titanium clip (Hemoclip®; Weck Closure System, Research Triangle Park, NC) with a defined internal diameter of 0.8 mm. After surgical closure of the thorax, the rats were allowed to recover from anesthesia. For postoperative analgesia, rats received 250 mg/kg bw of metamizole (Novalgin®; Aventis Pharma, Bad Sodem, Germany) intramuscularly immediately after the operation and on the first postoperative day. Sham-operated rats (i.e. , without insertion of a clip) served as controls. After recovery from anesthesia, the animals were placed in cages with free access to water and standard laboratory diet.

Nine weeks after aortic banding, rats had attained body weights of 360 ± 10 g and were anesthetized by intraperitoneal administration of urethane (0.15 g/100 g bw; Fluka, Buchs, Switzerland), followed by intramuscular injection of ketamine (0.05 mg/100 g bw; Pharmacia GmbH) as previously described.15The neck and chest wall were shaved, and rats were placed in the supine position on a thermostatically controlled electric heating blanket (Homeothermic Blanket Control Unit; Harvard Apparatus, March-Hugstetten, Germany) to maintain body temperature at 38°C during the experimental protocol. After tracheostomy, rats were mechanically ventilated (Rodent Respirator 680; Harvard Apparatus) with a tidal volume of 6 ml/kg bw at 110 breaths/min and a peak inspiratory pressure of 10.5 ± 1 cm H²O. Polyvinyl catheters (internal diameter 0.58 mm; Sims Portex Ltd., Hythe, United Kingdom) were introduced into the aorta and the vena cava via  the left carotid artery and the right jugular vein, respectively. A median thoracotomy was performed, and catheters were surgically placed into the left atrium and the pulmonary artery via  the left auricle and the right ventricle, respectively. An ultrasonic flowprobe (Transonic®; Transonic Systems Inc., Ithaca, NY) was positioned around the ascending aorta distal to the branching of the coronary arteries and proximal to the truncus brachiocephalicus. Arterial pressure (AP), central venous pressure (CVP), PAP, left atrial pressure (LAP), and airway pressure as well as aortic flow were continuously monitored and registered on-line with use of the software package DasyLab®32 (DasyLab, Moenchengladbach, Germany). SVR and PVR were calculated using standard equations.16 

For inhalation, milrinone (0.2–5 mg/ml, Corotrop®; Sanofi Winthrop, Paris, France) or NaCl (0.9%) were nebulized using an ultrasonic nebulizer (Optineb®; Nebu-Tec, Elsenfeld, Germany) and inhaled for 3 min at identical peak inspiratory pressures as used throughout the experiment. A 3-min nebulization of 1 mg/ml milrinone resulted in vaporization of 14 μg of the phosphodiesterase-3 inhibitor as determined by microgravimetry. Therefore, the respective dose of 39 μg/kg is analog to inhaled doses in human studies.10For intravenous delivery, milrinone (initial bolus of 2–10 μg/kg, followed by 0.2–1 μg · kg⁻¹· min⁻¹) or equivalent volumes of NaCl (0.9%; initial bolus of 1.6 ml/kg, followed by 10 ml · kg⁻¹· h⁻¹) were administered by an infusion pump (Perfusor® fm; B. Braun Melsungen AG, Melsungen, Germany) for 10 min.

Alveolar delivery of aerosolized substances was tested by inhalation of nebulized fluorescein isothiocyanate dextran (molecular weight 150 kd; Sigma-Aldrich, Taufkirchen, Germany) instead of milrinone and subsequent fluorescence imaging of subpleural alveoli in situ  as previously described.17In brief, after inhalation of fluorescein isothiocyanate dextran or NaCl (0.9%), lungs were excised, inflated with room air at 5 cm H²O, and imaged at λ= 480 nm (Axiotech^vario100HD; Carl Zeiss, Jena, Germany).

Rats were anesthetized and allowed to breathe spontaneously in the half left-lateral position. The long axis of the left ventricle was imaged by a 15-MHz transducer (SONOS 5500; Philips Medizin Systeme GmbH, Hamburg, Germany) as previously described.18From the two-dimensional mode of the long axis, end-diastolic volume (LVEDV) was determined. M-mode recordings from the short axis of the left ventricle were traced to yield ejection fraction, fractional shortening, and the wall thickness of the interventricular septum during diastole. Echocardiographic parameters were measured before and 1 min after milrinone inhalation.

Blood samples (1.5 ml) were obtained before and after inhalation of milrinone in sham-operated and CHF rats. Samples were collected in EDTA tubes, and plasma phosphodiesterases were immediately blocked by addition of IBMX (33 mg/ml; Sigma-Aldrich). After centrifugation at 4°C (5,000g  for 10 min), plasma aliquots were stored at −80°C and analyzed for cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) concentrations by enzyme-linked immunosorbent assay microplate immunoassay kits (R&D Systems, Minneapolis, MN).

Congestive heart failure rats were randomized into six groups as specified in table 1, groups i–vi. Hemodynamic measurements were performed before and after drug administration as illustrated by the flowchart of the experimental protocol (fig. 1A).

Animals were randomized into six groups and received inhalation (3 min) or intravenous (10 min) administration of either milrinone or NaCl as specified in table 1, groups 1–6. Blood samples for determination of cyclic nucleotides and mixed venous oxygen saturation (Svo²) were obtained and replaced by infusion of hydroxyethyl starch (6% hydroxyethyl starch 200/0.5; Fresenius, Bad Homburg, Germany) as illustrated in figure 1A. Hemodynamic parameters were recorded before and after drug administration. At the end of experiments, animals were killed by exsanguination, and the heart and lungs were removed. Hearts were dissected into the free wall of the right ventricle and the left ventricle including the septum and both atria and weighed. Lung wet/dry weight ratio was determined by use of the microwave drying technique.19 

Congestive heart failure rats were randomized into three groups (table 1, groups A–C) receiving none, one single, or three repeated milrinone inhalations for 3 min each in 20-min intervals over 1 h as illustrated in figure 1B. Hemodynamic parameters were continuously monitored and recorded every 10 min for 1 h, and lung wet/dry weight ratio was measured at the end of the experiments.

All data are presented as mean ± SEM. Values were compared by Wilcoxon or Friedman tests for intragroup data and by Kruskal–Wallis or Mann–Whitney U tests for intergroup data. Statistical significance was assumed at P < 0.05.

Within 9 weeks after supracoronary aortic banding, rats developed biventricular cardiac hypertrophy as demonstrated by increased heart/body weight ratio and ventricular masses in comparison with sham-operated animals (table 2). Increased hydrostatic pressures in the left atrium and the pulmonary artery demonstrate the development of CHF. Because the absolute increase in PAP exceeded the increase in LAP, both transpulmonary pressure gradient and PVR were increased, indicating increased pulmonary vascular tone or remodeling. AP, CVP, aortic flow, and systemic vascular resistance (SVR) in CHF rats did not differ from sham-operated animals demonstrating the absence of systemic hemodynamic effects.

Alveolar delivery of nebulized drugs was tested by inhalation of aerosolized fluorescein isothiocyanate dextran for 3 min. Subsequent fluorescence imaging showed strong and homogeneous fluorescence in all imaged alveoli, whereas no alveolar fluorescence was detected following inhalation of 0.9% NaCl (data not shown).

To establish dose–response relations for hemodynamic effects of milrinone in CHF, the effects of different drug concentrations delivered either by inhalation or by intravenous infusion on AP and PAP were tested. Inhalation of 1 mg/ml milrinone, a dose previously used in postcardiac surgical patients,10resulted in a near-maximal reduction of PAP without significant effects on AP (fig. 2A) and was therefore used subsequently for inhalation treatment. The reduction in PAP was less pronounced (P < 0.05) in response to the maximal intravenous dose tested, 1 μg · kg⁻¹· min⁻¹(fig. 2B), which to our knowledge is the highest dose reported for in vivo  use so far.20Lower intravenous doses had a lesser effect on PAP without improving pulmonary selectivity of the vasodilatory response. Therefore, a concentration of 1 μg · kg⁻¹· min⁻¹was subsequently used for intravenous delivery.

Inhalation of 1 mg/ml milrinone decreased pulmonary artery pressure similarly in a larger collective of CHF rats (fig. 3A). This effect was not attributable to milrinone effects on aortic flow, i.e. , cardiac output, but on a decrease in PVR by 39.9 ± 1.31% (P < 0.05) indicating pulmonary vasodilation. In addition, a small but significant decrease in LAP by 30.14 ± 2.29% was registered. AP, CVP, and SVR did not change (data not shown), whereas the PVR/SVR ratio decreased (fig. 3A), demonstrating that inhaled milrinone acted predominantly on the pulmonary circulation. Milrinone inhalation increased Svo²from 33 ± 1 to 42 ± 1%, but without reaching significance (P = 0.09). These changes were attributable to milrinone and not the inhalation procedure per se , because NaCl inhalation had no similar effects.

In sham-operated rats, inhalation of 1 mg/ml milrinone neither lowered PAP nor changed any of the other hemodynamic parameters, demonstrating the absence of pulmonary or systemic vascular effects in normal rats (fig. 3B).

In CHF rats, PAP (fig. 3C), PVR (−18.96 ± 1.7%), and LAP (−26.03 ± 2.3%) were also significantly reduced by intravenous infusion of 1 μg · kg⁻¹· min⁻¹milrinone (P < 0.05). However, intravenous milrinone also caused systemic vasodilation as evident from decreases in AP (fig. 3C) and SVR (−22.23 ± 1.6%). As a result, intravenous milrinone neither reduced the PVR/SVR ratio nor markedly improved Svo ²(37 ± 2% after milrinone vs.  33 ± 2% at baseline; P = 0.35).

After a single milrinone inhalation, PAP reached its minimum after 20 min and then slowly returned to baseline values (fig. 4). To test whether repeated milrinone inhalations may cause sustained pulmonary vasodilation, CHF rats inhaled milrinone in 20-min intervals, which produced a stable reduction of PAP over 60 min (fig. 4).

Potential propagation of hydrostatic lung edema is a major concern for the use of vasodilators in pulmonary venous hypertension. Analysis of wet/dry weight ratios showed a small but significant increase in CHF rats as compared with sham-operated animals (table 3). However, wet/dry weight ratio was not further increased 15 min after milrinone inhalation or infusion or even 60 min after inhalation of a single milrinone dose. Repeated milrinone inhalations even reduced lung wet/dry weight ratio, indicating that inhaled vasodilators may diminish lung edema in pulmonary venous hypertension.

In accordance with the characteristics of compensated left heart failure, CHF rats had a higher LVEDV and a thickening of the interventricular septum as compared with control animals, but did not differ with respect to ejection fraction or fractional shortening (table 4). Milrinone inhalation did not cause a detectable change in the dynamic geometry of the left ventricle and septum.

Whereas baseline cAMP concentrations were identical in CHF rats and control animals (fig. 5A), baseline cGMP levels were significantly lower in CHF rats (fig. 5B). Milrinone inhalation selectively increased cAMP but not cGMP plasma concentrations in both groups.

In a rat model of CHF, the phosphodiesterase-3 inhibitor milrinone reduced pulmonary hypertension and thus right ventricular afterload. Whereas intravenous infusion of milrinone resulted in systemic vasodilation, the action of inhaled milrinone was predominantly confined to the pulmonary vasculature. A sustained reduction of PAP was achieved by repeated milrinone inhalations. No hemodynamic indications of left ventricular volume overload were detected after milrinone inhalation. Edema formation was not amplified, but even reduced by repeated milrinone administrations. Therefore, inhaled milrinone may present an effective and safe new treatment strategy in pulmonary venous hypertension to unload the right ventricle and reduce edema formation.

In the current study, we determined the effects of milrinone in a rat model of CHF induced by supracoronary aortic banding,14an experimental model that reflects the pathophysiologic characteristics of compensated CHF due to supracoronary aortic stenosis in humans. Analogous to this clinical picture, increased hydrostatic pressure proximal to the stenosis resulted in left ventricular dilation and hypertrophy, whereas distally measured APs were unchanged. Unaltered left ventricular ejection fraction, fractional shortening, and cardiac output indicate the compensated state of CHF in this model. It should be considered that cardiovascular responses to inhaled or intravenous inodilators may differ in heart failure of alternative etiology such as ischemic heart disease. Because APs were determined distal to the aortic banding site, they do not adequately reflect left ventricular afterload and underestimate SVR by a systematic factor. However, aside from the constant resistance imposed by the aortic band, they yield reliable information on systemic vasomotor responses to intravenous or inhaled vasodilators. Alveolar deposition of the inhaled substances depends on size, charge, and lipophilia. Effective alveolar delivery of nebulized fluorescein isothiocyanate dextran, which exceeds milrinone in molecular weight by a factor of 700, was demonstrated by fluorescence imaging. Physiologic markers such as reduced PVR and increased cAMP plasma levels further confirm the effective delivery of milrinone by inhalation.

Endothelial dysfunction, i.e. , an imbalanced release of vasodilatory and vasoconstrictive factors in the lung resulting in pulmonary vasoconstriction and vascular remodeling, has been proposed to aggravate pulmonary hypertension in CHF.3Our findings support this concept because reduced cGMP levels measured in plasma of rats with supracoronary aortic banding indicate a deficiency of the endogenous nitric oxide–guanylate cyclase pathway. Consistent with the functional effects of reduced nitric oxide bioavailability, CHF rats had increased transpulmonary pressure gradients and an increased PVR. Inhaled or intravenous vasodilators rapidly reduced PVR to values similar to those measured in sham-operated rats. Because vascular remodeling cannot be reversed within this short time interval, these findings indicate that PVR elevation in CHF resulted predominantly from increased vascular tone. In sham-operated rats, vasodilator therapy had no pulmonary hemodynamic effects, which is in agreement with the notion that muscularized pulmonary vessels have only negligible tone under physiologic conditions.21 

The vasodilatory phosphodiesterase-3 inhibitor milrinone induces vessel relaxation22and increases myocardial contractility23by increasing the intracellular concentration of cAMP in smooth muscle cells and cardiomyocytes and has been approved for short-term intravenous treatment of patients with acute decompensated heart failure.24Here, milrinone given either by intravenous infusion or by inhalation effectively reduced PVR in CHF rats. In addition, a small decrease in LAP contributed to the pulmonary antihypertensive effect of both treatment strategies. The latter finding suggests additional cardiac effects of milrinone, presumably by improving left ventricular contractility and relaxation.25The fact that the positive inotropic effect of milrinone is not reflected by changes in systemic blood flow and AP is likely due to the mechanical outflow obstruction in the aortic banding model. Right-to-left ventricular septal wall interactions may provide an alternative explanation because reduction of right ventricular afterload may decompress the left ventricle. But echocardiographic data do not support this notion, because LVEDV and ejection fraction did not increase with milrinone inhalation in CHF rats.

Pulmonary vasodilation by intravenous infusion of milrinone was dose-dependent and paralleled by a marked hypotensive response in the systemic circulation. Previously, it had been hypothesized that systemic vasodilation may be beneficial in CHF by reducing left ventricular afterload. However, several clinical trials investigating the effects of the calcium channel blocker nifedipine,26oral milrinone,8the arteriovenous dilator flosequinan,27or systemic infusion of the prostacyclin analog epoprostenol (Flolan®; GlaxoSmithKline, Research Triangle Park, NC)9revealed that systemic vasodilator therapy in CHF patients is detrimental in terms of clinical outcome. The reasons underlying the negative outcome of these clinical trials are complex and incompletely understood. Besides potential drug interactions and inadequate data on pharmacokinetics, pharmacodynamics, and dose–response relations,27adverse effects of systemic vasodilation may aggravate the deterioration of right ventricular contractility, e.g. , by reducing coronary perfusion pressure.28 

Inhaled vasodilators may potentially circumvent these detrimental effects by acting predominantly in the pulmonary circulation with little or no vasodilatory effect in systemic blood vessels. Inhalation of 1 mg/ml milrinone reduced PAP more effectively than the maximal intravenous dose of 1 μg · kg⁻¹· min⁻¹, but without exerting systemic hemodynamic effects. A marked trend toward an increase in Svo²in the inhalation group but not in the intravenous group further suggests a beneficial potential for inhaled milrinone. The pulmonary vasodilatory effect of milrinone reached its maximum 20 min after inhalation and returned to a half-maximal effect after approximately 60 min, which is in general agreement with previously reported time profiles.10,29Repeated inhalation of milrinone in 20-min intervals warranted a stable reduction of PAP over the observation period of 1 h, demonstrating the potential of this strategy for prolonged treatment in CHF. Further studies in chronically instrumented animals are required to study the effects of long-term treatment by inhaled milrinone and potential rebound phenomena during subsequent weaning.

Major concerns in the treatment of CHF with vasodilators focus on the potential progression of lung edema and left ventricular dysfunction. The so-called Kitajew reflex, a precapillary pulmonary vasoconstrictive response to increased lung capillary pressure, has been postulated to protect the pulmonary capillary bed from excessive pressure increases in pulmonary venous hypertension.30Accordingly, vasodilator therapy may cause hydrostatic lung edema in pulmonary venous hypertension. In the current study, we detected a small but significant increase of lung wet/dry weight ratio in CHF rats as compared with sham-operated animals, indicating moderate lung edema, but this was not further aggravated by a single milrinone inhalation. This is in accordance with our findings that inhaled milrinone reduced PAP and vascular resistance in parallel without changing pulmonary flow, so that capillary pressure is likely to remain constant. However, precapillary vasoconstriction may constitute a protective mechanism when cardiac output is increased, and future studies are required to address the safety of inhaled vasodilators during exercise or β¹-adrenergic stimulation. Repeated milrinone inhalations even reduced lung wet/dry weight ratios, indicating an additional antiedematous effect. This finding may be attributable to decreased capillary filtration due to a reduction in LAP or to a cAMP-mediated enhancement of lung microvascular barrier function.31,32Vasodilator therapy has also been proposed to increase left ventricular filling pressure due to an increased venous return to a poorly compliant left ventricle in CHF.33However, our data do not support this concept, because milrinone inhalation reduced LAP and lung wet/dry weight ratios without affecting LVEDV or ejection fraction in CHF rats.

Caution must be exercised when transferring findings from animal models to the clinical situation. However, similar pathophysiologic mechanisms and the results of two small nonrandomized trials suggest that inhaled milrinone may also reduce right ventricular afterload in CHF patients. Endothelial dysfunction and increased PVR are characteristics of CHF not only in rats, but also in humans.3,34In nine cardiac surgical patients in the immediate postoperative period and in nine heart transplant candidates, inhaled milrinone was shown to induce a vasodilatory effect in the lung in the absence of systemic hemodynamic effects.10,29Consistent with the results of our study, inhaled milrinone did not exhibit adverse effects in CHF patients because it reduced pulmonary capillary wedge pressure and increased neither intrathoracic blood volume nor extravascular lung water.29Therefore, inhaled milrinone may present an effective and safe clinical strategy for the treatment of pulmonary venous hypertension due to CHF.

The authors thank Sylvia May (Technician, Institute of Physiology, Charité-Universitätsmedizin Berlin, Berlin, Germany) for excellent technical assistance.