Cardiovascular Drugs and Therapy 1992;6:67-75 © Kluwer Academic Publishers, Boston. Printed in U.S.A.
Beneficial Actions of Amlodipine in the Multiple-Stunned Canine Myocardium Garrett J. Gross a n d Galen M. P i e p e r Department of Pharmacologyand Toxicology, The Medical College of Wisconsin,Milwaukee, Wisconsin
Summary. The effects of the long-acting dihydropyridine calcium-entry blocker, amlodipine, on subendocardial segment shortening (%SS), regional myocardial blood flow (radioactive microspheres), and tissue high-energy phosphate levels were compared with those of a saline-treated group of barbital-anesthetized dogs subjected to nine 5-minute coronary artery occlusions interspersed with 15 minutes of reperfusion and finally by 1 hour of reperfusion (multiple stunned myocardium). Saline or amlodipine (200 ~g/kg, IV) were administered 15 minutes prior to the first coronary occlusion. There were no major differences between groups in ischemic bed size or hemoydnamics throughout the experiment. Subendocardial collateral blood flow was significantly increased in the amlodipine-treated group during coronary occlusion 1; however,tissue blood flow in the ischemic region was not significantly different between groups during occlusion 9. Following each occlusion, %SS in the ischemic region was equally reduced in both groups and passive systolic lengthening resulted. In spite of similar decreases in %SS during occlusion, the amlodipine-treated dogs showed a marked improvement in myocardial segment function (%SS) of the ischemic-reperfused region at 15 minutes following each occlusion (1-9) and at 15, 30, and 60 minutes of reperfusion following occlusion 9, as compared to saline-treated animals. In addition, amlodipine attenuated the loss of adenine nucleotides in the ischemic-reperfused area at 1 hour of reperfusion. These results suggest that amlodipine has a favorable effect on the functional and metabolic recovery of the multiple-stunned myocardium and may have potential as a cardioprotective agent for the treatment of myocardial reperfusion injury.
Cardiovasc Drugs Ther 1992;6:67-75 Key
Words. amlodipine, multiple-stunned myocardium, high-energy phosphates, collateral blood flow, reperfusion injury, calcium antagonist
Single or multiple, brief periods of coronary artery occlusion followed by reperfusion have been shown to result in prolonged, reversible decreases in regional contractile function and decreases in high-energy phosphates, a condition termed stunned or multiplestunned myocardium [1]. The primary mechanisms responsible for myocardial stunning in a single occlusion model are thought to be related to an increase in oxygen-derived free radicals or an interference with normal calcium homeostasis [1]. Marban et al. [2] sug-
gested that stunned myocardium most likely results from a decreased sensitivity of the myofilaments to cytosolic free calcium following reperfusion. This decreased myofilament sensitivity is thought to be the result of a transient calcium overload, which occurs during ischemia or early reperfusion [3]. A number of studies that demonstrate a favorable effect of calcium antagonists administered prior to occlusion or reperfusion favors the calcium overload hypothesis [4-6]. On the other hand, less work has been performed in elucidating the mechanism of stunning in the multiple occlusion model, although recent evidence exists to suggest an important role for oxygen-derived free radicals [7]. Reifart and coworkers [8] showed that two calcium antagonists, bepridil and verapamil, increased regional segment shortening in a canine model of multiple coronary occlusions and reperfusions, which suggests that calcium overload may also occur in this model. Thus, to further test the hypothesis that a transient calcium overload might contribute to the contractile dysfunction following multiple, brief periods of coronary artery occlusion, amlodipine, a long-acting, 1,4 dihydropyridine calcium antagonist (Figure 1), was administered prior to nine 5-minute coronary artery occlusions interspersed with 15 minutes of reperfusion and finally followed by 60 minutes of reperfusion. A dose (200 ~g/kg) of amlodipine was chosen that we previously had shown to enhance the recovery of systolic wall function in a single occlusion model where a mixture of reversible and irreversibly damaged tissue existed [9]. The multiple stunned model was chosen, since this model demonstrates only reversible injury [7,8] and is clinically relevant to the situation in unstable angina or coronary spasm where patients are subjected to multiple periods of occlusion and reperfusion [1]. Methods Adult mongrel dogs (18-30 kg) of either sex were anesthetized with sodium pentobarbital (15 mg/kg, IV) Address for correspondenceand reprint requests: Garrett J. Gross, Ph.D., Department of Pharmacologyand Toxicology,The Medical College of Wisconsin, 8701 WatertownPlank Road, Milwaukee,WI 53226. 67
68
Grossand Pieper
Cl H
H3C00C
C00C2H5
H3C'~ ~ N / ' C H 2 O C H 2 C H 2 N H 2 I H
I AmlodipineI Fig. 1. The che'mical structure of amlodipine.
and barbital sodium (200 mg/kg, IV), and were ventilated with a Harvard respirator (tidal volume of 15 ml/kg, 10-15 breaths/min) with room air supplemented with 100% 02. Atelectasis was prevented by maintaining an end expiratory pressure of 5-7 cm of water with a trap. Blood-gas samples were obtained from the right femoral artery and were maintained within the normal physiological range. Blood pH, Pco.- and Po2 were determined by a blood-gas analyzer (Radiometer ABL 2). Body temperature was maintained at 38°C with a heating pad. A double-tipped pressure transducer catheter (Millar PC 771) was inserted into the aorta and left ventricle via the carotid artery to monitor the mean aortic and left ventricular pressures. The left ventricular pressure pulse was electronically differentiated to obtain left ventricular dP/dt. The right femoral vein was cannulated for the administration of drug, vehicle, or subsequent anesthesia as needed. A left thoracotomy was performed at the fifth intercostal space, the lungs were retracted, the pericardium was incised, and the heart was suspended in a cradle. A 1.0- to 1.5-cm segment of the left anterior descending (LAD) coronary artery was dissected free from surrounding tissue distal to the first diagonal branch, and a calibrated electromagnetic flow probe (Statham SP 7515) was placed around the vessel. Coronary blood flow was measured with a flowmeter (Statham 2202). A micrometer-driven mechanical occluder was placed distal to the flow probe such that there were no branches between the probe and occluder. The occluder was used to zero the flow probe and later to occlude the artery. Heart rate was monitored using limb lead I I from the electrocardiogram and a tachograph (Grass Model 7P4F). All hemodynamics were monitored on a Grass model 7 polygraph.
Myocardial s e g m e n t shortening Myocardial segment function was measured in the regions perfused by the LAD and left circumflex artery (LC) by two sets of piezoelectric crystals inserted 7-9 mm into the subendocardium. Crystal depths were verified at the end of each experiment. The leads of the crystals were connected to an ultrasonic amplifier, which transforms the crystal-transmitted sound pulse into an electrical signal proportional to the distance between them. The tracings were monitored with an oscilloscope (Soltec Model 520). The distance between the two crystals was measured by recording changes in the transmission time. Diastolic segment length (DL) was determined at the beginning of the rise phase of positive dP/dt (onset of isovolumetric contraction), and systolic segment length (SL) was determined at the peak negative dP/dt. Percent segment shortening (%SS) was calculated using the equation %SS = (DL - SL)/DL × 100. The segment length data was normalized by using a value of 10.0 for the control DL.
Myocardial blood flow Transmural myocardial blood flow was determined by the radioactive microsphere technique (15 ~Lm spheres). Briefly, 10-20 ~LCi of either raCe, l°~Ru, or 9~Nb (approximately 2-4 x 106 spheres) were injected into the left atrium followed by a 6-ml saline flush. Prior to microsphere administration, a collection of reference blood flow from the right femoral artery was begun at the rate of 6.8 ml/min and was maintained for 3 minutes. At the completion of each experiment, India ink was injected into the LAD at the point of the flow probe in order to delineate the ischemic perfusion area. Left ventricular weights, ischemic area weights, and areas at risk were similar in both groups (Figure 2). The heart was subsequently removed and stored overnight in 10% formalin. The heart was sectioned into tissue pieces from the ischemic [5] and nonischemic [3] areas. Only pieces within at least 2 cm of the black-dyed area were included in the data analysis. The pieces were each sectioned into subepicardium, midmyocardium, and subendocardium. Tissue and reference blood flow samples were counted in a gamma counter (Searle Analytic 1195). Myocardial blood flow was calculated using a preprogrammed computer (Apple IIe) to obtain the true activity of each isotope in individual samples. Tissue blood flow was calculated by the following equation: Qm = Qr.Cm/Cr, where Qm is myocardial blood flow (ml/min/g), Qr is the rate of withdrawal of the reference blood flow sample (cpm), and Cm is the activity of the tissue sample (cpm/g). Transmural blood flow was calculated as the weighted average of the three layers in each
A m l o d i p i n e a n d Multiple S t u n n e d M y o c a r d i u m
• CONTROL
,50[
0
[ ] AMLODIPINE{200J~g/kg)
LV Wt. (g)
Isch.Wt. (g)
Risk Area (%)
Fig. 2. Left ventricular weight ( L V Wt), ischemic area weight (Isch Wt), a n d the risk area as a percent of L V Wt i~t the co~trol ( N = 9) a n d amlodopine-treated ( N - 8) gro~tps. A l l values are the m e a n +_ S E M .
region. The mean endocardial/epicardial (endo/epi) blood flow ratio was also determined.
Myocardial biopsies and metabolism At the completion of each experiment (60 minutes after the final reperfusion), a small area of the normal and ischemic region was painted with methylene blue dye. Transmural drill biopsies were obtained from normal and ischemic areas at the dye site and were frozen in liquid nitrogen as described previously [10]. The frozen biopsy was divided into three approximately equal transmural sections: epicardium, midmyocardium, and endocardium. The frozen sections were weighed and homogenized in 6% perchloric acid using a Tekmar tissue homogenizer. Extracts were neutralized with 5 M K2CO3 and the supernatant was used for biochemical analyses. A 100 mm 3 aliquot of neutralized extract was used in a coupled enzymatic reaction to determine phosphocreatine (PCr) and ATP. In a separate reaction, ADP and AMP w e r e determined [10]. A portion of tissue from each layer was also dried to a constant weight at 95°C in pretared beakers for wet/dry weight calculations. All metabolic data w e r e expressed in units of ~Lmol/g dry tissue weight. The total adenine nucleotide pool was also calculated (ATP + ADP + AMP).
Experimental protocol The experimental design included a pretreatmentcontrol measurement of hemodynamics, myocardial segment function, and blood flow following instrumen-
69
tation of the animal. Microspheres were administered before saline or drug intervention. Animals were randomized into two groups. Either saline (control series) or amlodipine (200 ~g/kg, IV) were administered over a 2-minute period via the right femoral vein 15 minutes prior to the first LAD occlusion. Ten minutes after drug or saline treatment, hemodynamics and myocardial segment function were determined. The LAD was then occluded for 5 minutes nine times with 15 minutes of reperfusion interspersed between each occlusion period (total occlusion period = 45 minutes), and hemodynamics and myocardial segment shortening were determined during occlusion and 15 minutes following each occlusion. At the end of the ninth occlusion period, the occluder was released and hemodynamics and myocardial function were determined at 15, 30, and 60 minutes of reperfusion. Radioactive microspheres were also administered during occlusion 1 and 9, and at 60 minutes of reperfusion, to determine regional myocardial blood flow.
Statistical analysis All values are the mean -+ SEM. Hemodynamics were obtained from a mean of 3-5 cardiac cycles. Groups w e r e compared using a two-way analysis of variance with repeated measures, and Fisher's least significant difference (LSD) was used to test for the significance of difference between any two groups at specific time points. When occlusion values were compared to the pretreatment control, Dunnet's test was used. Means w e r e considered significantly different if p < 0.05.
Results Hemodynamics Hemodynamics during preocclusion, occlusions 1 and 9, and at 15, 30, or 60 minutes ofreperfusion following occlusion 9 are shown in Table 1. In the control series, no significant hemodynamic changes occurred throughout the experiment. In amlodipine-treated animals, there were significant decreases in mean aortic blood pressure and left ventricular systolic pressure only during the first occlusion period, as compared to the preocclusion control value and the corresponding value in the control series. However, no differences in hemodynamics between groups were observed throughout the remainder of the experiment.
Regional myocardial blood flow Tissue blood-flow data during preocclusion, occlusions 1 and 9, and at 60 minutes of reperfusion following occlusion 9 are summarized in Tables 2 and 3. No changes were observed in the nonischemic left circumflex (LC) region in the control series (Table 2) throughout the experiment. In the amlodipine-treated dogs, there was an increase in flow to all layers of the nonischemic area throughout occlusion and r e p e r f u -
70
Grossand Pieper
Table 1. Hemodynamics in the control and amlodipine-treated animals
Preocclusion Control Amlodipine Occlusion 1 Control Amlodipme Occlusion 9 Control Amlodipme Reperfusion 9 (15 rain) Control Amlodipme Reperfusion 9 (30 min) Control Amlodipme Reperfusion 9 (60 rain) Control Amlodipme All values are the mean -+ SEM HR = heart rate; LVSP = left aSigniflcantly different from the bSignificantly different from the
H e a r t rate (beats/rain)
Mean aortic pressure (mmHg)
Left ventricular systolic p r e s s u r e (mmHg)
+ dP/dtma x (mmHg/sec)
HR x LVSP (mmHg/min/104)
142±6 147±6
110±6 107±6
125±7 121±8
2450 ± 221 2475 ± 223
1.81 ± 0.16 1.79 -+ 0.15
114±5 101 ± 7 a.b
2214 ± 194 2297 ± 210
1.65 ± 0.13 1.45 ± 0.14
143±6 141 ± 5
99±5 86 ± 5 a,b
144-+5 143 ± 6
105 ± 6 104 ± 4
122±7 121 ± 5
2467 ± 173 2597 ± 208
1.76 ± 0.14 1.74 ± 0.12
139-+5 139±6
112±5 108-+4
130±6 125 ± 4
2417 ± 104 2634 ± 191
1.79 ± 0.10 1.74 ± 0.11
137±5 144 ± 6
110±6 109± 6
127-+8 127± 5
2317 ± 175 2719 ± 167
1.74 ± 0.13 1.83 ± 0.13
138±6 141 ± 6
114±7 110-+5
132±8 127 ± 5
2367 ± 87 2588 ± 200
1.81 ± 0.13 1.80 ± 0.13
(n = 9, control group; n = 8, amlodipine group). ventricular systolic pressure. preocclusion control value within each group (p < 0.05). corresponding value in the control group (p < 0.05).
Table 2. Myocardial tissue blood flow in normal and ischemic-reperfused regions in the control series Preocclusion
Occlusion 1
Occlusion 9
1 hour reperfusion
1.16 1.25 1.49 1.30 1.30
± ± ± ±
0.13 0.17 0.18 0.16 0.05
1.02 1.11 1.30 1.14 1.27
± ± ± ± ±
0.11 0.15 0.15 0.14 0.05
1.03 1.05 1.16 1.08 1.14
± ± ± ± ±
0.07 0.08 0.08 0.07 0.05
1.13 1.14 1.27 1.18 1.15
± ± ± ± ±
0.15 0.17 0.17 0.16 0.06
1.16 1.22 1.23 1.21 1.10
± -± ± ±
0.15 0.12 0.15 0.13 0.09
0.25 0.15 0.12 0.17 0.50
± ± ± ± -+
0.05 b 0.03 b 0.02 b 0.03 b 0.10 b
0.32 0.16 0.14 0.20 0.46
-+ 0.08 b ± 0.04 b ± 0.03 b --- 0.05 b -+ 0.09 b
1.35 0.92 0.89 1.06 0.71
± ± ± ± ±
0.17 0.10 a 0.11 ~ 0.12 0.08 a
Normal LC region Epicardial Midmyoeardial Endocardial Transmural Endo/Epi
Ischemic LAD region Epicardial Midmyocardial Endocardial Transmural Endo/Epi
All values (ml/min/g) are the mean -+ SEM LC = left circumflex; LAD = left anterior ap < 0.05 versus preocclusion control value bp < 0.01 versus preocclusion control value
sion; however, subepicardium
(n = 9). descending. within each group. within each group.
this increase was only significant in the during the first occlusion period (Table
3). In the ischemic-reperfused LAD region, coronary occlusion produced marked decreases in transmural b l o o d f l o w i n b o t h s e r i e s o f a n i m a l s ( T a b l e s 2 a n d 3). T h e r e w a s n o d i f f e r e n c e i n c o l l a t e r a l b l o o d f l o w in all
layers of the ischemic region during occlusions 1 and 9, w i t h t h e e x c e p t i o n o f a g r e a t e r s u b e n d o c a r d i a l blood flow in the amlodipine series during occlusion 1 ( F i g u r e 3). A t 1 h o u r o f r e p e r f u s i o n , m i d m y o c a r d i a l and subendocardial flows were significantly lower t h a n p r e o c c l u s i o n v a l u e s in t h e c o n t r o l s e r i e s ( T a b l e 2), b u t n o t i n a m l o d i p i n e - t r e a t e d d o g s ( T a b l e 3).
Amlodipine and Multiple Stunned Myocardium
71
Table 3. Myocardial tissue blood flow in normal and ischemic-reperfused regions in the amlodipine series Preocclusion
Occlusion 1
Occlusion 9
1 hour reperfusion
1.04 1.12 1.28 1.15 1.24
+ 0.13 + 0.14 _+ 0.15 -+ 0.13 -+ 0.03
1.81 1.81 1.91 1.85 1.04
-+ 0.25 a -+ 0.27 -+ 0.39 -+ 0.29 _+ 0.13
1.54 1.41 1.47 1.47 0.99
-+ 0.20 _+ 0.19 _+ 0.19 _+ 0.18 -+ 0.11
1.71 1.50 1.79 1.67 1.06
_+ 0.31 -+ 0.27 -+ 0.32 -+ 0.30 -+ 0.08
1.12 1.09 1.12 1.11 1.01
-+ -+ -+ -+ -+
0.33 0.17 0.24 0.24 0.78
-+ -+ -+ -+ -+
0.38 0.18 0.23 0.26 0.80
-+ -+ -+ -+ +-
1.46 1.09 1.19 1.25 0.80
-+ 0.28 -+ 0.24 _+ 0.27 -+ 0.26 -+ 0.10
Normal LC region Epicardial Midmyocardial Endocardial Transmural Endo/Epi
Ischemic L A D region Epicardial Midmyocardial Endocardial Transmural Endo/Epi
All values (ml/min/g) are the m e a n -+ SEM LC = left circumflex; L A D = left anterior ap < 0.05 v e r s u s preocclusion control value bp < 0.01 v e r s u s preocclusion control value
0.15 0.15 0.16 0.14 0.08
0.06 b 0.03 b 0.06 b 0.05 b 0.16
0.09 b 0.045 0.04 b 0.055 0.20
( n = 8). descending. within each group. within each group.
AMLOOIPINE
'•E 0.6
N.S.]
~0.4
i
0
Epi I
Mid Occlusion (1)
Enclo
Epi
I
[
N.S.
I
Mid
Endo
Occlusion (9)
J
Fig. 3. Collateral blood.flow (ml/min/g) in the subepicardium (Epi), midmyocardium (Mid), and subendocardium (Endo) during occlusions I and 9 in the control (N = 9) and amlodipine-treated (N = 8) groups. All values are the mea~l +_ SEM. *Significantly different f r o m the control group (p < 0.05). N.S. = not significant.
Myocardial segment function Myocardial segment shortening (%SS) data are summarized in Figures 4 and 5. No significant changes in %SS occurred in the nonischemic LC area in either series throughout the experiment (data not shown). In the ischemic-reperfused LAD region, coronary occlusion resulted in an equivalent reduction in %SS to negative values in both groups during occlusion 1 and 9 (see Figure 5), which is indicative of passive systolic lengthening or bulging during ischemia.
At 15 minutes of reperfusion following each occlusion period (1-9), the amlodipine-treated dogs had a significantly greater %SS than at the corresponding time in the control series (Figure 4). During the last 60 minutes of reperfusion, %SS in the control and amlodipine-treated groups was decreased from the preocclusion values; however, in the amlodipinetreated dogs the recovery of %SS was significantly greater at all times as compared to the control group (Figure 5).
72
Grossand Pieper
I H CONTROL ] o--o AMLODIPINE(200ug/kg) .
.
~"
';IAT
.
A h'~.OOIPINE
"~
1 oo
• P
Beneficial Actions of Amlodipine in the Multiple-Stunned Canine Myocardium Garrett J. Gross a n d Galen M. P i e p e r Department of Pharmacologyand Toxicology, The Medical College of Wisconsin,Milwaukee, Wisconsin
Summary. The effects of the long-acting dihydropyridine calcium-entry blocker, amlodipine, on subendocardial segment shortening (%SS), regional myocardial blood flow (radioactive microspheres), and tissue high-energy phosphate levels were compared with those of a saline-treated group of barbital-anesthetized dogs subjected to nine 5-minute coronary artery occlusions interspersed with 15 minutes of reperfusion and finally by 1 hour of reperfusion (multiple stunned myocardium). Saline or amlodipine (200 ~g/kg, IV) were administered 15 minutes prior to the first coronary occlusion. There were no major differences between groups in ischemic bed size or hemoydnamics throughout the experiment. Subendocardial collateral blood flow was significantly increased in the amlodipine-treated group during coronary occlusion 1; however,tissue blood flow in the ischemic region was not significantly different between groups during occlusion 9. Following each occlusion, %SS in the ischemic region was equally reduced in both groups and passive systolic lengthening resulted. In spite of similar decreases in %SS during occlusion, the amlodipine-treated dogs showed a marked improvement in myocardial segment function (%SS) of the ischemic-reperfused region at 15 minutes following each occlusion (1-9) and at 15, 30, and 60 minutes of reperfusion following occlusion 9, as compared to saline-treated animals. In addition, amlodipine attenuated the loss of adenine nucleotides in the ischemic-reperfused area at 1 hour of reperfusion. These results suggest that amlodipine has a favorable effect on the functional and metabolic recovery of the multiple-stunned myocardium and may have potential as a cardioprotective agent for the treatment of myocardial reperfusion injury.
Cardiovasc Drugs Ther 1992;6:67-75 Key
Words. amlodipine, multiple-stunned myocardium, high-energy phosphates, collateral blood flow, reperfusion injury, calcium antagonist
Single or multiple, brief periods of coronary artery occlusion followed by reperfusion have been shown to result in prolonged, reversible decreases in regional contractile function and decreases in high-energy phosphates, a condition termed stunned or multiplestunned myocardium [1]. The primary mechanisms responsible for myocardial stunning in a single occlusion model are thought to be related to an increase in oxygen-derived free radicals or an interference with normal calcium homeostasis [1]. Marban et al. [2] sug-
gested that stunned myocardium most likely results from a decreased sensitivity of the myofilaments to cytosolic free calcium following reperfusion. This decreased myofilament sensitivity is thought to be the result of a transient calcium overload, which occurs during ischemia or early reperfusion [3]. A number of studies that demonstrate a favorable effect of calcium antagonists administered prior to occlusion or reperfusion favors the calcium overload hypothesis [4-6]. On the other hand, less work has been performed in elucidating the mechanism of stunning in the multiple occlusion model, although recent evidence exists to suggest an important role for oxygen-derived free radicals [7]. Reifart and coworkers [8] showed that two calcium antagonists, bepridil and verapamil, increased regional segment shortening in a canine model of multiple coronary occlusions and reperfusions, which suggests that calcium overload may also occur in this model. Thus, to further test the hypothesis that a transient calcium overload might contribute to the contractile dysfunction following multiple, brief periods of coronary artery occlusion, amlodipine, a long-acting, 1,4 dihydropyridine calcium antagonist (Figure 1), was administered prior to nine 5-minute coronary artery occlusions interspersed with 15 minutes of reperfusion and finally followed by 60 minutes of reperfusion. A dose (200 ~g/kg) of amlodipine was chosen that we previously had shown to enhance the recovery of systolic wall function in a single occlusion model where a mixture of reversible and irreversibly damaged tissue existed [9]. The multiple stunned model was chosen, since this model demonstrates only reversible injury [7,8] and is clinically relevant to the situation in unstable angina or coronary spasm where patients are subjected to multiple periods of occlusion and reperfusion [1]. Methods Adult mongrel dogs (18-30 kg) of either sex were anesthetized with sodium pentobarbital (15 mg/kg, IV) Address for correspondenceand reprint requests: Garrett J. Gross, Ph.D., Department of Pharmacologyand Toxicology,The Medical College of Wisconsin, 8701 WatertownPlank Road, Milwaukee,WI 53226. 67
68
Grossand Pieper
Cl H
H3C00C
C00C2H5
H3C'~ ~ N / ' C H 2 O C H 2 C H 2 N H 2 I H
I AmlodipineI Fig. 1. The che'mical structure of amlodipine.
and barbital sodium (200 mg/kg, IV), and were ventilated with a Harvard respirator (tidal volume of 15 ml/kg, 10-15 breaths/min) with room air supplemented with 100% 02. Atelectasis was prevented by maintaining an end expiratory pressure of 5-7 cm of water with a trap. Blood-gas samples were obtained from the right femoral artery and were maintained within the normal physiological range. Blood pH, Pco.- and Po2 were determined by a blood-gas analyzer (Radiometer ABL 2). Body temperature was maintained at 38°C with a heating pad. A double-tipped pressure transducer catheter (Millar PC 771) was inserted into the aorta and left ventricle via the carotid artery to monitor the mean aortic and left ventricular pressures. The left ventricular pressure pulse was electronically differentiated to obtain left ventricular dP/dt. The right femoral vein was cannulated for the administration of drug, vehicle, or subsequent anesthesia as needed. A left thoracotomy was performed at the fifth intercostal space, the lungs were retracted, the pericardium was incised, and the heart was suspended in a cradle. A 1.0- to 1.5-cm segment of the left anterior descending (LAD) coronary artery was dissected free from surrounding tissue distal to the first diagonal branch, and a calibrated electromagnetic flow probe (Statham SP 7515) was placed around the vessel. Coronary blood flow was measured with a flowmeter (Statham 2202). A micrometer-driven mechanical occluder was placed distal to the flow probe such that there were no branches between the probe and occluder. The occluder was used to zero the flow probe and later to occlude the artery. Heart rate was monitored using limb lead I I from the electrocardiogram and a tachograph (Grass Model 7P4F). All hemodynamics were monitored on a Grass model 7 polygraph.
Myocardial s e g m e n t shortening Myocardial segment function was measured in the regions perfused by the LAD and left circumflex artery (LC) by two sets of piezoelectric crystals inserted 7-9 mm into the subendocardium. Crystal depths were verified at the end of each experiment. The leads of the crystals were connected to an ultrasonic amplifier, which transforms the crystal-transmitted sound pulse into an electrical signal proportional to the distance between them. The tracings were monitored with an oscilloscope (Soltec Model 520). The distance between the two crystals was measured by recording changes in the transmission time. Diastolic segment length (DL) was determined at the beginning of the rise phase of positive dP/dt (onset of isovolumetric contraction), and systolic segment length (SL) was determined at the peak negative dP/dt. Percent segment shortening (%SS) was calculated using the equation %SS = (DL - SL)/DL × 100. The segment length data was normalized by using a value of 10.0 for the control DL.
Myocardial blood flow Transmural myocardial blood flow was determined by the radioactive microsphere technique (15 ~Lm spheres). Briefly, 10-20 ~LCi of either raCe, l°~Ru, or 9~Nb (approximately 2-4 x 106 spheres) were injected into the left atrium followed by a 6-ml saline flush. Prior to microsphere administration, a collection of reference blood flow from the right femoral artery was begun at the rate of 6.8 ml/min and was maintained for 3 minutes. At the completion of each experiment, India ink was injected into the LAD at the point of the flow probe in order to delineate the ischemic perfusion area. Left ventricular weights, ischemic area weights, and areas at risk were similar in both groups (Figure 2). The heart was subsequently removed and stored overnight in 10% formalin. The heart was sectioned into tissue pieces from the ischemic [5] and nonischemic [3] areas. Only pieces within at least 2 cm of the black-dyed area were included in the data analysis. The pieces were each sectioned into subepicardium, midmyocardium, and subendocardium. Tissue and reference blood flow samples were counted in a gamma counter (Searle Analytic 1195). Myocardial blood flow was calculated using a preprogrammed computer (Apple IIe) to obtain the true activity of each isotope in individual samples. Tissue blood flow was calculated by the following equation: Qm = Qr.Cm/Cr, where Qm is myocardial blood flow (ml/min/g), Qr is the rate of withdrawal of the reference blood flow sample (cpm), and Cm is the activity of the tissue sample (cpm/g). Transmural blood flow was calculated as the weighted average of the three layers in each
A m l o d i p i n e a n d Multiple S t u n n e d M y o c a r d i u m
• CONTROL
,50[
0
[ ] AMLODIPINE{200J~g/kg)
LV Wt. (g)
Isch.Wt. (g)
Risk Area (%)
Fig. 2. Left ventricular weight ( L V Wt), ischemic area weight (Isch Wt), a n d the risk area as a percent of L V Wt i~t the co~trol ( N = 9) a n d amlodopine-treated ( N - 8) gro~tps. A l l values are the m e a n +_ S E M .
region. The mean endocardial/epicardial (endo/epi) blood flow ratio was also determined.
Myocardial biopsies and metabolism At the completion of each experiment (60 minutes after the final reperfusion), a small area of the normal and ischemic region was painted with methylene blue dye. Transmural drill biopsies were obtained from normal and ischemic areas at the dye site and were frozen in liquid nitrogen as described previously [10]. The frozen biopsy was divided into three approximately equal transmural sections: epicardium, midmyocardium, and endocardium. The frozen sections were weighed and homogenized in 6% perchloric acid using a Tekmar tissue homogenizer. Extracts were neutralized with 5 M K2CO3 and the supernatant was used for biochemical analyses. A 100 mm 3 aliquot of neutralized extract was used in a coupled enzymatic reaction to determine phosphocreatine (PCr) and ATP. In a separate reaction, ADP and AMP w e r e determined [10]. A portion of tissue from each layer was also dried to a constant weight at 95°C in pretared beakers for wet/dry weight calculations. All metabolic data w e r e expressed in units of ~Lmol/g dry tissue weight. The total adenine nucleotide pool was also calculated (ATP + ADP + AMP).
Experimental protocol The experimental design included a pretreatmentcontrol measurement of hemodynamics, myocardial segment function, and blood flow following instrumen-
69
tation of the animal. Microspheres were administered before saline or drug intervention. Animals were randomized into two groups. Either saline (control series) or amlodipine (200 ~g/kg, IV) were administered over a 2-minute period via the right femoral vein 15 minutes prior to the first LAD occlusion. Ten minutes after drug or saline treatment, hemodynamics and myocardial segment function were determined. The LAD was then occluded for 5 minutes nine times with 15 minutes of reperfusion interspersed between each occlusion period (total occlusion period = 45 minutes), and hemodynamics and myocardial segment shortening were determined during occlusion and 15 minutes following each occlusion. At the end of the ninth occlusion period, the occluder was released and hemodynamics and myocardial function were determined at 15, 30, and 60 minutes of reperfusion. Radioactive microspheres were also administered during occlusion 1 and 9, and at 60 minutes of reperfusion, to determine regional myocardial blood flow.
Statistical analysis All values are the mean -+ SEM. Hemodynamics were obtained from a mean of 3-5 cardiac cycles. Groups w e r e compared using a two-way analysis of variance with repeated measures, and Fisher's least significant difference (LSD) was used to test for the significance of difference between any two groups at specific time points. When occlusion values were compared to the pretreatment control, Dunnet's test was used. Means w e r e considered significantly different if p < 0.05.
Results Hemodynamics Hemodynamics during preocclusion, occlusions 1 and 9, and at 15, 30, or 60 minutes ofreperfusion following occlusion 9 are shown in Table 1. In the control series, no significant hemodynamic changes occurred throughout the experiment. In amlodipine-treated animals, there were significant decreases in mean aortic blood pressure and left ventricular systolic pressure only during the first occlusion period, as compared to the preocclusion control value and the corresponding value in the control series. However, no differences in hemodynamics between groups were observed throughout the remainder of the experiment.
Regional myocardial blood flow Tissue blood-flow data during preocclusion, occlusions 1 and 9, and at 60 minutes of reperfusion following occlusion 9 are summarized in Tables 2 and 3. No changes were observed in the nonischemic left circumflex (LC) region in the control series (Table 2) throughout the experiment. In the amlodipine-treated dogs, there was an increase in flow to all layers of the nonischemic area throughout occlusion and r e p e r f u -
70
Grossand Pieper
Table 1. Hemodynamics in the control and amlodipine-treated animals
Preocclusion Control Amlodipine Occlusion 1 Control Amlodipme Occlusion 9 Control Amlodipme Reperfusion 9 (15 rain) Control Amlodipme Reperfusion 9 (30 min) Control Amlodipme Reperfusion 9 (60 rain) Control Amlodipme All values are the mean -+ SEM HR = heart rate; LVSP = left aSigniflcantly different from the bSignificantly different from the
H e a r t rate (beats/rain)
Mean aortic pressure (mmHg)
Left ventricular systolic p r e s s u r e (mmHg)
+ dP/dtma x (mmHg/sec)
HR x LVSP (mmHg/min/104)
142±6 147±6
110±6 107±6
125±7 121±8
2450 ± 221 2475 ± 223
1.81 ± 0.16 1.79 -+ 0.15
114±5 101 ± 7 a.b
2214 ± 194 2297 ± 210
1.65 ± 0.13 1.45 ± 0.14
143±6 141 ± 5
99±5 86 ± 5 a,b
144-+5 143 ± 6
105 ± 6 104 ± 4
122±7 121 ± 5
2467 ± 173 2597 ± 208
1.76 ± 0.14 1.74 ± 0.12
139-+5 139±6
112±5 108-+4
130±6 125 ± 4
2417 ± 104 2634 ± 191
1.79 ± 0.10 1.74 ± 0.11
137±5 144 ± 6
110±6 109± 6
127-+8 127± 5
2317 ± 175 2719 ± 167
1.74 ± 0.13 1.83 ± 0.13
138±6 141 ± 6
114±7 110-+5
132±8 127 ± 5
2367 ± 87 2588 ± 200
1.81 ± 0.13 1.80 ± 0.13
(n = 9, control group; n = 8, amlodipine group). ventricular systolic pressure. preocclusion control value within each group (p < 0.05). corresponding value in the control group (p < 0.05).
Table 2. Myocardial tissue blood flow in normal and ischemic-reperfused regions in the control series Preocclusion
Occlusion 1
Occlusion 9
1 hour reperfusion
1.16 1.25 1.49 1.30 1.30
± ± ± ±
0.13 0.17 0.18 0.16 0.05
1.02 1.11 1.30 1.14 1.27
± ± ± ± ±
0.11 0.15 0.15 0.14 0.05
1.03 1.05 1.16 1.08 1.14
± ± ± ± ±
0.07 0.08 0.08 0.07 0.05
1.13 1.14 1.27 1.18 1.15
± ± ± ± ±
0.15 0.17 0.17 0.16 0.06
1.16 1.22 1.23 1.21 1.10
± -± ± ±
0.15 0.12 0.15 0.13 0.09
0.25 0.15 0.12 0.17 0.50
± ± ± ± -+
0.05 b 0.03 b 0.02 b 0.03 b 0.10 b
0.32 0.16 0.14 0.20 0.46
-+ 0.08 b ± 0.04 b ± 0.03 b --- 0.05 b -+ 0.09 b
1.35 0.92 0.89 1.06 0.71
± ± ± ± ±
0.17 0.10 a 0.11 ~ 0.12 0.08 a
Normal LC region Epicardial Midmyoeardial Endocardial Transmural Endo/Epi
Ischemic LAD region Epicardial Midmyocardial Endocardial Transmural Endo/Epi
All values (ml/min/g) are the mean -+ SEM LC = left circumflex; LAD = left anterior ap < 0.05 versus preocclusion control value bp < 0.01 versus preocclusion control value
sion; however, subepicardium
(n = 9). descending. within each group. within each group.
this increase was only significant in the during the first occlusion period (Table
3). In the ischemic-reperfused LAD region, coronary occlusion produced marked decreases in transmural b l o o d f l o w i n b o t h s e r i e s o f a n i m a l s ( T a b l e s 2 a n d 3). T h e r e w a s n o d i f f e r e n c e i n c o l l a t e r a l b l o o d f l o w in all
layers of the ischemic region during occlusions 1 and 9, w i t h t h e e x c e p t i o n o f a g r e a t e r s u b e n d o c a r d i a l blood flow in the amlodipine series during occlusion 1 ( F i g u r e 3). A t 1 h o u r o f r e p e r f u s i o n , m i d m y o c a r d i a l and subendocardial flows were significantly lower t h a n p r e o c c l u s i o n v a l u e s in t h e c o n t r o l s e r i e s ( T a b l e 2), b u t n o t i n a m l o d i p i n e - t r e a t e d d o g s ( T a b l e 3).
Amlodipine and Multiple Stunned Myocardium
71
Table 3. Myocardial tissue blood flow in normal and ischemic-reperfused regions in the amlodipine series Preocclusion
Occlusion 1
Occlusion 9
1 hour reperfusion
1.04 1.12 1.28 1.15 1.24
+ 0.13 + 0.14 _+ 0.15 -+ 0.13 -+ 0.03
1.81 1.81 1.91 1.85 1.04
-+ 0.25 a -+ 0.27 -+ 0.39 -+ 0.29 _+ 0.13
1.54 1.41 1.47 1.47 0.99
-+ 0.20 _+ 0.19 _+ 0.19 _+ 0.18 -+ 0.11
1.71 1.50 1.79 1.67 1.06
_+ 0.31 -+ 0.27 -+ 0.32 -+ 0.30 -+ 0.08
1.12 1.09 1.12 1.11 1.01
-+ -+ -+ -+ -+
0.33 0.17 0.24 0.24 0.78
-+ -+ -+ -+ -+
0.38 0.18 0.23 0.26 0.80
-+ -+ -+ -+ +-
1.46 1.09 1.19 1.25 0.80
-+ 0.28 -+ 0.24 _+ 0.27 -+ 0.26 -+ 0.10
Normal LC region Epicardial Midmyocardial Endocardial Transmural Endo/Epi
Ischemic L A D region Epicardial Midmyocardial Endocardial Transmural Endo/Epi
All values (ml/min/g) are the m e a n -+ SEM LC = left circumflex; L A D = left anterior ap < 0.05 v e r s u s preocclusion control value bp < 0.01 v e r s u s preocclusion control value
0.15 0.15 0.16 0.14 0.08
0.06 b 0.03 b 0.06 b 0.05 b 0.16
0.09 b 0.045 0.04 b 0.055 0.20
( n = 8). descending. within each group. within each group.
AMLOOIPINE
'•E 0.6
N.S.]
~0.4
i
0
Epi I
Mid Occlusion (1)
Enclo
Epi
I
[
N.S.
I
Mid
Endo
Occlusion (9)
J
Fig. 3. Collateral blood.flow (ml/min/g) in the subepicardium (Epi), midmyocardium (Mid), and subendocardium (Endo) during occlusions I and 9 in the control (N = 9) and amlodipine-treated (N = 8) groups. All values are the mea~l +_ SEM. *Significantly different f r o m the control group (p < 0.05). N.S. = not significant.
Myocardial segment function Myocardial segment shortening (%SS) data are summarized in Figures 4 and 5. No significant changes in %SS occurred in the nonischemic LC area in either series throughout the experiment (data not shown). In the ischemic-reperfused LAD region, coronary occlusion resulted in an equivalent reduction in %SS to negative values in both groups during occlusion 1 and 9 (see Figure 5), which is indicative of passive systolic lengthening or bulging during ischemia.
At 15 minutes of reperfusion following each occlusion period (1-9), the amlodipine-treated dogs had a significantly greater %SS than at the corresponding time in the control series (Figure 4). During the last 60 minutes of reperfusion, %SS in the control and amlodipine-treated groups was decreased from the preocclusion values; however, in the amlodipinetreated dogs the recovery of %SS was significantly greater at all times as compared to the control group (Figure 5).
72
Grossand Pieper
I H CONTROL ] o--o AMLODIPINE(200ug/kg) .
.
~"
';IAT
.
A h'~.OOIPINE
"~
1 oo
• P
