European Journal of Pharmacology, 182 (1990) 527-535 Elsevier
527
EJP 51399
Beneficial hemodynamic effects of two weeks' milrinone treatment in conscious rats with heart failure Regien G. Schoemaker, Jacques J.M. Debets, Harry A.J. Struyker-Boudier and Jos F.M. Smits Department of Pharmacology, University of Limburg~ Maastricht, The Netherlands Received 18 January 1990, revised MS received 9 March 1990, accepted 17 April 1990
Milrinone is a phosphodiesterase inhibitor which combines vasodilating effects with inotropic effects. Hemodynamic improvement after acute administration and increased survival with chronic milrinone therapy in rats with heart failure have been reported before, and suggest long-term hemodynamic improvement. However, no detailed hemodynamic studies are available on prolonged milrinone therapy in rats with heart failure. Therefore, the hemodynamic effects of 2 weeks' milrinone therapy were now investigated in conscious rats with heart failure due to myocardial infarction. The effects were compared to hemodynamic changes after acute administration. Acute milrinone increased the baseline cardiac output in infarcted rats by increasing heart rate rather than stroke volume. However, the maximal cardiac output achieved when the heart was stimulated through a volume load was improved due to increased stroke volume as well as increased heart rate. The increase in maximally stimulated cardiac output after acute milrinone was found to be related to infarct size. Two weeks' milrinone therapy in chronically infarcted rats dose dependently restored the hemodynamic changes which were caused by infarction. In contrast to acute administration, two weeks' milrinone restored cardiac function without an increase in heart rate. The effects were achieved at a rate of administration which presumably has no acute inotropic effects. The data indicate that acute milrinone in infarcted rats has vasodilating effects. Positive inotropic effects, possibly masked by concomitant venodilatation at baseline conditions, became overt after stimulation by volume loading. Long-term milrinone dose dependently restored cardiac function in infarcted rats without effects on heart rate or mean arterial pressure, suggesting that different mechanisms may be involved. Milrinone; Heart failure; Hemodynamics; Myocardial infarct; (Rat)
I. Introduction T h e r a p y o f h e a r t failure i n c l u d e s the use of positive i n o t r o p i c a n d v a s o d i l a t i n g drugs. M i l r i n o n e is a p h o s p h o d i e s t e r a s e i n h i b i t o r w i t h p o s i t i v e i n o t r o p i c as well as v a s o d i l a t i n g p r o p e r t i e s in m a n .
J.F.M. Smits is an Established Investigator of the Dutch Heart Foundation. Correspondence to: R.G. Schoemaker, Department of Pharmacology, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
W h e r e a s there is g e n e r a l a g r e e m e n t a b o u t the hem o d y n a m i c i m p r o v e m e n t after a c u t e a d m i n i s t r a tion ( B a l m et al., 1983; Sys et al., 1986; Piscione et al., 1987; Evans, 1989), t h e r e are conflicting rep o r t s a b o u t p r o g r e s s i o n o f the disease a n d m o r t a l ity ( M o n r a d et al., 1986; B a i m et al., 1986; L e J e m t e l et al., 1989). This m a y b e c a u s e d b y differences b e t w e e n p a t i e n t s , i n t e r f e r e n c e w i t h add i t i o n a l t h e r a p y o r d e s i g n of the studies. A large l o n g - t e r m m i l r i n o n e survival s t u d y has recently b e e n a n n o u n c e d (Packer, 1989). T h e results after 3 m o n t h s o f t h e r a p y , however, i n d i c a t e i n c r e a s e d r a t h e r t h a n r e d u c e d m o r t a l i t y ( D i B i a n c o et al., 1989).
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
528
Milrinone has also been reported to have acute positive inotropic and vasodilating effects in infarcted rats (Emmert et al., 1987; Drexler et al., 1987; DeFelice et al., 1989). Moreover, milrinone not only improves hemodynamics after acute therapy, but also increases survival on chronic therapy of infarcted rats (Sweet et al., 1988). This suggests a long-term hemodynamic improvement. However, no detailed hemodynami c studies with prolonged milrinone in rats with heart failure are available. Therefore, the hemodynamic effects of 2 weeks' milrinone as monotherapy were now investigated in conscious rats with heart failure after myocardial infarction. The effects were compared to the hemodynamic changes after acute administration to control rats and to rats with heart failure. To avoid interference with the repair processes after myocardial infarction (Fishbein et al., 1978; Pfeffer et al., 1979) the protocols were started at least 3 weeks after ligation.
2. Materials and methods
Male Wistar rats (Winkelman, Borchem, FRG), weighing 332 + 6 g at the start of the protocol, were used throughout this study. After surgery, the animals were housed separately in macrolon cages on sawdust in air-conditioned rooms with a 12-h light-dark cycle. The animals had free access to standard lab food (Hope Farms, Woerden, The Netherlands) and tap water before and during all experiments. All experiments were performed in conformity with the ethical codes of the University of Limburg, The Netherlands.
2.1. Coronary artery ligation Ligation of the left descending coronary artery of the rats was done as described in detail elsewhere (Fishbein et al., 1978; Pfeffer et al., 1979; Schoemaker et al., in press). The rats were anesthetized with pentobarbital (60 m g / k g i.p.) and the trachea was intubated. Skin and muscles overlying the 4th left intercostal space were separated and cut. Lidocaine, 2 m g / k g i.m., was given prophylactically (Botting et al., 1983; Leprhn
et al., 1983). The thorax was opened after starting positive pressure respiration and the heart was carefully pushed to the left or exteriorized by applying pressure to the right side of the thorax. A 6-0 silk suture was looped under the left coronary artery near the origin of the pulmonary artery. In sham-operated rats the ligature was looped through the myocardium right next to the coronary artery. The suture was tied after the heart had been returned to its normal position. The ribs were pulled together with 3-0 silk, muscles and skin were sutured separately and subatmospheric pressure was restored by inserting a silastic drain through a stab wound between the 6th and 7th rib and application of a negative pressure of 10 cm H20.
2.2. Preparation for hemodynamic measurements One week before hemodynamic measurements an electromagnetic flow probe (2.6-2.7 mm, Skalar, Delft, The Netherlands) was implanted on the ascending aorta through a right thoracotomy as described in detail elsewhere (Smits et al., 1982). The connector was exteriorized in the neck and fixed to the skin. Subatmospheric pressure was again restored through a drain. The animals were allowed 5-6 days to recover from this surgery. Following the recovery period, the animals were anesthetized with ether and a catheter (PE-10 heat-sealed to PE-50) was inserted into the abdominal aorta through a femoral artery for measurement of arterial blood pressure. Three catheters were introduced into the vena cava through the femoral veins. The first two (PE-10 heat-sealed to PE-50) were inserted up to approximately 4 cm, their tips being located in the abdominal vena cava. The third venous catheter (silastic 602-175) was advanced into the femoral vein up to about 9 cm, which located its tip in the thoracic vena cava near the right atrium. The first catheters were used for infusion, whereas the latter was used to measure central venous pressure. All catheters were filled with normal saline, closed with metal plugs and exteriorized in the neck. Two subcutaneous electrodes (teflon-coated silver, 0.25 mm) were implanted, one over the sacral region and the other over the right scapula. These elec-
529 trodes, soldered to a miniature connector exteriorized in the neck, were used to record the ECG (Jensen et al., 1984). Following this surgery, the animals were allowed at least 1 day for recovery.
2.3. Protocol for acute milrinone administration The protocol was started three weeks after ligation. Hemodynamic measurements were performed every other day in order to avoid any effects of a previous volume load or drug infusion. The rats were subjected in random order to control volume loading or volume loading during the infusion of 30 # g / k g per min milrinone (Schoemaker et al., 1990). The animals were connected to the measuring equipment: a sine-wave electromagnetic flowmeter (Skalar, Delft, The Netherlands), miniature lowvolume displacement pressure transducers (CP-01, Century Technology Company, Inglewood, Ca, USA) and an E C G amplifier. The signals were fed into a 68B09-based pre-processor and an AT-compatible microcomputer for on-line derivation of hemodynamic parameters. Cardiac output (CO), heart rate (HR), aortic flow acceleration ( d F / d t ) , peak flow (PF), stroke volume (SV) and duration of the flow pulse (left ventricular ejection time, LVET), were derived from the flow signal. Mean arterial pressure (MAP) and central venous pressure (CVP) were recorded and the resting respiration rate was determined. Stroke work (SW) was computed as SW = SV* ( M A P - CVP). Total peripheral resistance (TPR) was computed as T P R = (MAP - CVP)/CO). The time period between the Q peak in the E C G and the start of the subsequent aortic flow pulse was taken as the pre-ejection period (PEP) and the ratio P E P / L V E T was computed. The data were displayed continuously and stored on a hard disk for later analysis. Equilibration for 45-60 mill was followed by measurement of baseline values for 15 min. Either a control volume load was then given or milrinone infusion was started (total volume infused < 1 ml). A volume load consisted of an i.v. infusion of 12 ml of warm (37 ° C) Ringer solution in 1 min. This volume load increased CO to a plateau level after about 45 s in spite of increasing CVP further
(Schoemaker et al., 1990). Hemodynamics were monitored continuously during this intervention and recording was continued for approximately 30 min after the infusion. In the case of milrinone infusion, the volume loading protocol as described above was repeated when the hemodynamics had again stabilized or at least 30 min after the start of milrinone infusion.
2. 4. Protocol for 2 weeks" milrinone therapy Three weeks after coronary artery ligation, the rats were divided into three groups. One group received no treatment and the other groups were treated with 30 or 150 # g / k g per h milrinone. Osmotic minipumps (Alzet 2001, ALZA, USA) filled with milrinone solution were implanted subcutaneously between the shoulder blades under light ether anesthesia. The pumps were replaced by new ones after 1 week and the electromagnetic flow probe was implanted at this time. Hemodynamic measurements were performed after 2 weeks' therapy. The rats were connected to the measuring equipment and signals were fed into the pre-processor and computer for on-line derivation of hemodynamic parameters. Baseline values were obtained after equilibration for 45-60 min. The heart was then stimulated by volume loading. Hemodynamics were monitored continuously until CVP had returned to baseline values or maximally 60 min after volume loading. The data were stored on a hard disk for off-line analysis.
2.5. Measurements of infarct size The animals were killed at the end of the experiments: the hearts were arrested in diastole with 1 M KC1 i.v. under pentobarbital anesthesia. The hearts were excised, washed in saline, vessels and atria were removed and the ventricles weighed. After weighing, the hearts were quickly frozen ( - 80 ° C ) to be cut into 1-mm slices from apex to base. The slices were stained with nitro blue tetrazolium (NBT), as described elsewhere (Leprhn et al., 1983; Schoemaker et al., in press). N B T stains all tissue vital at the time of death, thus gives no information about the area at risk. Color pictures were taken and the lengths of the infarcted area
530 m i n i s t r a t i o n were c o m p a r e d , using S t u d e n t ' s t-test for p a i r e d o b s e r v a t i o n s . T h e d a t a are e x p r e s s e d as m e a n s + S.E.M., unless i n d i c a t e d otherwise. Differences o r correlations were t a k e n to b e statistically significant if P < 0.05.
a n d of the s p a r e d m u s c l e were d e t e r m i n e d b y p l a n i m e t r y for b o t h the e n d o c a r d i a l a n d e p i c a r d i a l surfaces o f each slice. T h e m e t h o d for these m e a s u r e m e n t s was d e s c r i b e d in detail b y Pfeffer et al. (1985). F i n a l i n f a r c t size was e x p r e s s e d as p e r c e n t of left v e n t r i c u l a r c i r c u m f e r e n c e c a l c u l a t e d as the average of i n f a r c t size of e n d o c a r d i a l a n d epicardial surfaces. T h e m e a n e n d o c a r d i a l c i r c u m f e r e n c e ( L V E C ) was used as an e s t i m a t e of left v e n t r i c u l a r dilatation.
3. Results
3.1. Coronary artery ligation 2.6. Data analysis M o r t a l i t y over the first 24 h after c o r o n a r y a r t e r y l i g a t i o n was 30%. I n a d d i t i o n , two rats d i e d b e t w e e n 1 d a y a n d 3 weeks after ligation. C o r o n a r y a r t e r y l i g a t i o n r e s u l t e d exclusively in i n f a r c t i o n s in t h e free w a l l o f the left ventricle. P a p i l l a r y muscles were u s u a l l y s p a r e d . I n f a r c t s were t r a n s m u r a l exc e p t for t h o s e s m a l l e r t h a n 10%. I n f a r c t sizes in surviving rats r a n g e d f r o m 13 to 54%. A t gross a u t o p s y at the e n d o f the e x p e r i m e n t s , m o s t of the i n f a r c t e d rats s h o w e d signs of c o n g e s t i o n in liver a n d lungs a n d h a d p l e u r a l effusate.
T h e results c o m p r i s e d a t a f r o m 7-13 a n i m a l s p e r g r o u p (cf. tables 1 a n d 2). T h e s h a m c o n t r o l g r o u p was c o m p o s e d of a n i m a l s which u n d e r w e n t the s a m e surgery a n d p r o t o c o l , received n o treatm e n t a n d h a d m e a s u r e d infarct sizes of 0% at the e n d of the e x p e r i m e n t . Overall c o m p a r i s o n s o f the e x p e r i m e n t a l g r o u p s were m a d e b y o n e - w a y analysis of v a r i a n c e ( A N O V A ) a n d a m o d i f i e d (Bonferroni) t-test ( W a l l e n s t e i n et al., 1980). T h e effects o f i n f a r c t i o n a n d 2 weeks' m i l f i n o n e t h e r a p y were c o m p a r e d b y o n e - w a y A N O V A a n d D u n n e t t ' s test. T h e correlations b e t w e e n h e m o d y n a m i c s a n d i n f a r c t sizes were a n a l y z e d b y l i n e a r regression analysis. H e m o d y n a m i c s b e f o r e a n d after a c u t e m i l r i n o n e a d m i n i s t r a t i o n , as well as c o n t r o l c a r d i a c f u n c t i o n a n d c a r d i a c f u n c t i o n d u r i n g a c u t e m i l r i n o n e ad-
3.2. Acute effects o f milrinone T h e effects o n b a s e l i n e h e m o d y n a m i c s a n d on c a r d i a c f u n c t i o n were s t u d i e d in i n f a r c t e d rats a n d in s h a m c o n t r o l rats. T h e results are s u m m a r i z e d in t a b l e 1. Baseline C O was slightly b u t signifi-
TABLE 1 Acute effects of 30/~g/kg-rain milrinone in infarcted (infarct size 32 ± 5%) and sham control rats. Baseline (c) and volume load VL (c) include values for control volume load, whereas baseline (d), drug (d) and volume load, VL (d), include baseline values before, baseline values during, and stimulated values during drug infusion respectively. Abbreviations: VL, volume load; MAP, mean arterial pressure (mmHg); HR, heart rate (b.min-l); SV, stroke volume (/~1); CO, cardiac output (ml.min-l). Volume load with milrinone
Control volume load
Control (n = 7) MAP HR
SV CO
Infarct (n = 13) MAP HR
SV CO
Baseline (c)
VL (c)
Baseline (d)
Drug (d)
VL (d)
112+ 5 386 ± 17 253 ± 17 95+ 4
114+ 5 400 ± 10 358 ± 11 141± 6
1055:5 385 ± 18 248 ± 18 96± 6
94+ 6 428 ± 19 236 + 23 104± 5 a
89-t- 5 b 425 ± 11 396 5:10 b 167± 5 b
96+ 3 368 + 11 210 + 12 765:4
90+ 4 398 + 5 287 + 18 1145:7
95+ 5 371 +
91+ 4 a
83+ 5
8
418 + 11 a
436 + 11 b
231 + 10 855:4
223 + 11 935:5 a
324 + 19 b 142+10 b
a Significant effect on baseline values, b Significant effect on maximally stimulated values.
531
r=-.927 (p
527
EJP 51399
Beneficial hemodynamic effects of two weeks' milrinone treatment in conscious rats with heart failure Regien G. Schoemaker, Jacques J.M. Debets, Harry A.J. Struyker-Boudier and Jos F.M. Smits Department of Pharmacology, University of Limburg~ Maastricht, The Netherlands Received 18 January 1990, revised MS received 9 March 1990, accepted 17 April 1990
Milrinone is a phosphodiesterase inhibitor which combines vasodilating effects with inotropic effects. Hemodynamic improvement after acute administration and increased survival with chronic milrinone therapy in rats with heart failure have been reported before, and suggest long-term hemodynamic improvement. However, no detailed hemodynamic studies are available on prolonged milrinone therapy in rats with heart failure. Therefore, the hemodynamic effects of 2 weeks' milrinone therapy were now investigated in conscious rats with heart failure due to myocardial infarction. The effects were compared to hemodynamic changes after acute administration. Acute milrinone increased the baseline cardiac output in infarcted rats by increasing heart rate rather than stroke volume. However, the maximal cardiac output achieved when the heart was stimulated through a volume load was improved due to increased stroke volume as well as increased heart rate. The increase in maximally stimulated cardiac output after acute milrinone was found to be related to infarct size. Two weeks' milrinone therapy in chronically infarcted rats dose dependently restored the hemodynamic changes which were caused by infarction. In contrast to acute administration, two weeks' milrinone restored cardiac function without an increase in heart rate. The effects were achieved at a rate of administration which presumably has no acute inotropic effects. The data indicate that acute milrinone in infarcted rats has vasodilating effects. Positive inotropic effects, possibly masked by concomitant venodilatation at baseline conditions, became overt after stimulation by volume loading. Long-term milrinone dose dependently restored cardiac function in infarcted rats without effects on heart rate or mean arterial pressure, suggesting that different mechanisms may be involved. Milrinone; Heart failure; Hemodynamics; Myocardial infarct; (Rat)
I. Introduction T h e r a p y o f h e a r t failure i n c l u d e s the use of positive i n o t r o p i c a n d v a s o d i l a t i n g drugs. M i l r i n o n e is a p h o s p h o d i e s t e r a s e i n h i b i t o r w i t h p o s i t i v e i n o t r o p i c as well as v a s o d i l a t i n g p r o p e r t i e s in m a n .
J.F.M. Smits is an Established Investigator of the Dutch Heart Foundation. Correspondence to: R.G. Schoemaker, Department of Pharmacology, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
W h e r e a s there is g e n e r a l a g r e e m e n t a b o u t the hem o d y n a m i c i m p r o v e m e n t after a c u t e a d m i n i s t r a tion ( B a l m et al., 1983; Sys et al., 1986; Piscione et al., 1987; Evans, 1989), t h e r e are conflicting rep o r t s a b o u t p r o g r e s s i o n o f the disease a n d m o r t a l ity ( M o n r a d et al., 1986; B a i m et al., 1986; L e J e m t e l et al., 1989). This m a y b e c a u s e d b y differences b e t w e e n p a t i e n t s , i n t e r f e r e n c e w i t h add i t i o n a l t h e r a p y o r d e s i g n of the studies. A large l o n g - t e r m m i l r i n o n e survival s t u d y has recently b e e n a n n o u n c e d (Packer, 1989). T h e results after 3 m o n t h s o f t h e r a p y , however, i n d i c a t e i n c r e a s e d r a t h e r t h a n r e d u c e d m o r t a l i t y ( D i B i a n c o et al., 1989).
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
528
Milrinone has also been reported to have acute positive inotropic and vasodilating effects in infarcted rats (Emmert et al., 1987; Drexler et al., 1987; DeFelice et al., 1989). Moreover, milrinone not only improves hemodynamics after acute therapy, but also increases survival on chronic therapy of infarcted rats (Sweet et al., 1988). This suggests a long-term hemodynamic improvement. However, no detailed hemodynami c studies with prolonged milrinone in rats with heart failure are available. Therefore, the hemodynamic effects of 2 weeks' milrinone as monotherapy were now investigated in conscious rats with heart failure after myocardial infarction. The effects were compared to the hemodynamic changes after acute administration to control rats and to rats with heart failure. To avoid interference with the repair processes after myocardial infarction (Fishbein et al., 1978; Pfeffer et al., 1979) the protocols were started at least 3 weeks after ligation.
2. Materials and methods
Male Wistar rats (Winkelman, Borchem, FRG), weighing 332 + 6 g at the start of the protocol, were used throughout this study. After surgery, the animals were housed separately in macrolon cages on sawdust in air-conditioned rooms with a 12-h light-dark cycle. The animals had free access to standard lab food (Hope Farms, Woerden, The Netherlands) and tap water before and during all experiments. All experiments were performed in conformity with the ethical codes of the University of Limburg, The Netherlands.
2.1. Coronary artery ligation Ligation of the left descending coronary artery of the rats was done as described in detail elsewhere (Fishbein et al., 1978; Pfeffer et al., 1979; Schoemaker et al., in press). The rats were anesthetized with pentobarbital (60 m g / k g i.p.) and the trachea was intubated. Skin and muscles overlying the 4th left intercostal space were separated and cut. Lidocaine, 2 m g / k g i.m., was given prophylactically (Botting et al., 1983; Leprhn
et al., 1983). The thorax was opened after starting positive pressure respiration and the heart was carefully pushed to the left or exteriorized by applying pressure to the right side of the thorax. A 6-0 silk suture was looped under the left coronary artery near the origin of the pulmonary artery. In sham-operated rats the ligature was looped through the myocardium right next to the coronary artery. The suture was tied after the heart had been returned to its normal position. The ribs were pulled together with 3-0 silk, muscles and skin were sutured separately and subatmospheric pressure was restored by inserting a silastic drain through a stab wound between the 6th and 7th rib and application of a negative pressure of 10 cm H20.
2.2. Preparation for hemodynamic measurements One week before hemodynamic measurements an electromagnetic flow probe (2.6-2.7 mm, Skalar, Delft, The Netherlands) was implanted on the ascending aorta through a right thoracotomy as described in detail elsewhere (Smits et al., 1982). The connector was exteriorized in the neck and fixed to the skin. Subatmospheric pressure was again restored through a drain. The animals were allowed 5-6 days to recover from this surgery. Following the recovery period, the animals were anesthetized with ether and a catheter (PE-10 heat-sealed to PE-50) was inserted into the abdominal aorta through a femoral artery for measurement of arterial blood pressure. Three catheters were introduced into the vena cava through the femoral veins. The first two (PE-10 heat-sealed to PE-50) were inserted up to approximately 4 cm, their tips being located in the abdominal vena cava. The third venous catheter (silastic 602-175) was advanced into the femoral vein up to about 9 cm, which located its tip in the thoracic vena cava near the right atrium. The first catheters were used for infusion, whereas the latter was used to measure central venous pressure. All catheters were filled with normal saline, closed with metal plugs and exteriorized in the neck. Two subcutaneous electrodes (teflon-coated silver, 0.25 mm) were implanted, one over the sacral region and the other over the right scapula. These elec-
529 trodes, soldered to a miniature connector exteriorized in the neck, were used to record the ECG (Jensen et al., 1984). Following this surgery, the animals were allowed at least 1 day for recovery.
2.3. Protocol for acute milrinone administration The protocol was started three weeks after ligation. Hemodynamic measurements were performed every other day in order to avoid any effects of a previous volume load or drug infusion. The rats were subjected in random order to control volume loading or volume loading during the infusion of 30 # g / k g per min milrinone (Schoemaker et al., 1990). The animals were connected to the measuring equipment: a sine-wave electromagnetic flowmeter (Skalar, Delft, The Netherlands), miniature lowvolume displacement pressure transducers (CP-01, Century Technology Company, Inglewood, Ca, USA) and an E C G amplifier. The signals were fed into a 68B09-based pre-processor and an AT-compatible microcomputer for on-line derivation of hemodynamic parameters. Cardiac output (CO), heart rate (HR), aortic flow acceleration ( d F / d t ) , peak flow (PF), stroke volume (SV) and duration of the flow pulse (left ventricular ejection time, LVET), were derived from the flow signal. Mean arterial pressure (MAP) and central venous pressure (CVP) were recorded and the resting respiration rate was determined. Stroke work (SW) was computed as SW = SV* ( M A P - CVP). Total peripheral resistance (TPR) was computed as T P R = (MAP - CVP)/CO). The time period between the Q peak in the E C G and the start of the subsequent aortic flow pulse was taken as the pre-ejection period (PEP) and the ratio P E P / L V E T was computed. The data were displayed continuously and stored on a hard disk for later analysis. Equilibration for 45-60 mill was followed by measurement of baseline values for 15 min. Either a control volume load was then given or milrinone infusion was started (total volume infused < 1 ml). A volume load consisted of an i.v. infusion of 12 ml of warm (37 ° C) Ringer solution in 1 min. This volume load increased CO to a plateau level after about 45 s in spite of increasing CVP further
(Schoemaker et al., 1990). Hemodynamics were monitored continuously during this intervention and recording was continued for approximately 30 min after the infusion. In the case of milrinone infusion, the volume loading protocol as described above was repeated when the hemodynamics had again stabilized or at least 30 min after the start of milrinone infusion.
2. 4. Protocol for 2 weeks" milrinone therapy Three weeks after coronary artery ligation, the rats were divided into three groups. One group received no treatment and the other groups were treated with 30 or 150 # g / k g per h milrinone. Osmotic minipumps (Alzet 2001, ALZA, USA) filled with milrinone solution were implanted subcutaneously between the shoulder blades under light ether anesthesia. The pumps were replaced by new ones after 1 week and the electromagnetic flow probe was implanted at this time. Hemodynamic measurements were performed after 2 weeks' therapy. The rats were connected to the measuring equipment and signals were fed into the pre-processor and computer for on-line derivation of hemodynamic parameters. Baseline values were obtained after equilibration for 45-60 min. The heart was then stimulated by volume loading. Hemodynamics were monitored continuously until CVP had returned to baseline values or maximally 60 min after volume loading. The data were stored on a hard disk for off-line analysis.
2.5. Measurements of infarct size The animals were killed at the end of the experiments: the hearts were arrested in diastole with 1 M KC1 i.v. under pentobarbital anesthesia. The hearts were excised, washed in saline, vessels and atria were removed and the ventricles weighed. After weighing, the hearts were quickly frozen ( - 80 ° C ) to be cut into 1-mm slices from apex to base. The slices were stained with nitro blue tetrazolium (NBT), as described elsewhere (Leprhn et al., 1983; Schoemaker et al., in press). N B T stains all tissue vital at the time of death, thus gives no information about the area at risk. Color pictures were taken and the lengths of the infarcted area
530 m i n i s t r a t i o n were c o m p a r e d , using S t u d e n t ' s t-test for p a i r e d o b s e r v a t i o n s . T h e d a t a are e x p r e s s e d as m e a n s + S.E.M., unless i n d i c a t e d otherwise. Differences o r correlations were t a k e n to b e statistically significant if P < 0.05.
a n d of the s p a r e d m u s c l e were d e t e r m i n e d b y p l a n i m e t r y for b o t h the e n d o c a r d i a l a n d e p i c a r d i a l surfaces o f each slice. T h e m e t h o d for these m e a s u r e m e n t s was d e s c r i b e d in detail b y Pfeffer et al. (1985). F i n a l i n f a r c t size was e x p r e s s e d as p e r c e n t of left v e n t r i c u l a r c i r c u m f e r e n c e c a l c u l a t e d as the average of i n f a r c t size of e n d o c a r d i a l a n d epicardial surfaces. T h e m e a n e n d o c a r d i a l c i r c u m f e r e n c e ( L V E C ) was used as an e s t i m a t e of left v e n t r i c u l a r dilatation.
3. Results
3.1. Coronary artery ligation 2.6. Data analysis M o r t a l i t y over the first 24 h after c o r o n a r y a r t e r y l i g a t i o n was 30%. I n a d d i t i o n , two rats d i e d b e t w e e n 1 d a y a n d 3 weeks after ligation. C o r o n a r y a r t e r y l i g a t i o n r e s u l t e d exclusively in i n f a r c t i o n s in t h e free w a l l o f the left ventricle. P a p i l l a r y muscles were u s u a l l y s p a r e d . I n f a r c t s were t r a n s m u r a l exc e p t for t h o s e s m a l l e r t h a n 10%. I n f a r c t sizes in surviving rats r a n g e d f r o m 13 to 54%. A t gross a u t o p s y at the e n d o f the e x p e r i m e n t s , m o s t of the i n f a r c t e d rats s h o w e d signs of c o n g e s t i o n in liver a n d lungs a n d h a d p l e u r a l effusate.
T h e results c o m p r i s e d a t a f r o m 7-13 a n i m a l s p e r g r o u p (cf. tables 1 a n d 2). T h e s h a m c o n t r o l g r o u p was c o m p o s e d of a n i m a l s which u n d e r w e n t the s a m e surgery a n d p r o t o c o l , received n o treatm e n t a n d h a d m e a s u r e d infarct sizes of 0% at the e n d of the e x p e r i m e n t . Overall c o m p a r i s o n s o f the e x p e r i m e n t a l g r o u p s were m a d e b y o n e - w a y analysis of v a r i a n c e ( A N O V A ) a n d a m o d i f i e d (Bonferroni) t-test ( W a l l e n s t e i n et al., 1980). T h e effects o f i n f a r c t i o n a n d 2 weeks' m i l f i n o n e t h e r a p y were c o m p a r e d b y o n e - w a y A N O V A a n d D u n n e t t ' s test. T h e correlations b e t w e e n h e m o d y n a m i c s a n d i n f a r c t sizes were a n a l y z e d b y l i n e a r regression analysis. H e m o d y n a m i c s b e f o r e a n d after a c u t e m i l r i n o n e a d m i n i s t r a t i o n , as well as c o n t r o l c a r d i a c f u n c t i o n a n d c a r d i a c f u n c t i o n d u r i n g a c u t e m i l r i n o n e ad-
3.2. Acute effects o f milrinone T h e effects o n b a s e l i n e h e m o d y n a m i c s a n d on c a r d i a c f u n c t i o n were s t u d i e d in i n f a r c t e d rats a n d in s h a m c o n t r o l rats. T h e results are s u m m a r i z e d in t a b l e 1. Baseline C O was slightly b u t signifi-
TABLE 1 Acute effects of 30/~g/kg-rain milrinone in infarcted (infarct size 32 ± 5%) and sham control rats. Baseline (c) and volume load VL (c) include values for control volume load, whereas baseline (d), drug (d) and volume load, VL (d), include baseline values before, baseline values during, and stimulated values during drug infusion respectively. Abbreviations: VL, volume load; MAP, mean arterial pressure (mmHg); HR, heart rate (b.min-l); SV, stroke volume (/~1); CO, cardiac output (ml.min-l). Volume load with milrinone
Control volume load
Control (n = 7) MAP HR
SV CO
Infarct (n = 13) MAP HR
SV CO
Baseline (c)
VL (c)
Baseline (d)
Drug (d)
VL (d)
112+ 5 386 ± 17 253 ± 17 95+ 4
114+ 5 400 ± 10 358 ± 11 141± 6
1055:5 385 ± 18 248 ± 18 96± 6
94+ 6 428 ± 19 236 + 23 104± 5 a
89-t- 5 b 425 ± 11 396 5:10 b 167± 5 b
96+ 3 368 + 11 210 + 12 765:4
90+ 4 398 + 5 287 + 18 1145:7
95+ 5 371 +
91+ 4 a
83+ 5
8
418 + 11 a
436 + 11 b
231 + 10 855:4
223 + 11 935:5 a
324 + 19 b 142+10 b
a Significant effect on baseline values, b Significant effect on maximally stimulated values.
531
r=-.927 (p
