On the Nature of Protection by Propranolol Against Myocardial Necrosis After Temporary Coronary Occlusion in Dogs
KEITH A. REIMER, MD, PhD MARGARET M. RASMUSSEN* ROBERT B. JENNINGS, MD
Propranolol has been shown to reduce the extent of necrosis that develops after temporary coronary occlusion in dogs. To determine whether this protective action was related to beta adrenergic block-
Chicago, Illinois
ade or to direct effects, necrosis was quantitated in the posterior papillary muscle 2 to 4 days after 40 minute periods of coronary occlusion in anesthetized open chest dogs. Groups of dogs either were untreated or were pretreated with doses of d,l-propranolol, 0.005 to 5 mg/kg body weight, or doses of d-propranolol 2.5 or 5 mg/kg. Necrosis was greatly reduced
in dogs treated
with 5 mg/kg
of d,l-propranol-
01. This protective effect was significant but quantitatively less with 0.5 and 0.05 mg/kg of d,l-propranolol. A dose of 0.005 mg/kg d,l-propranolol and d-propranolol failed to alter myocardial necrosis significantly. The dose-related reduction of necrosis with d,l-propranolol correlated with a similar dose relation for beta adrenergic blockade and suggested that a protective effect was related to beta blockade. The reduction of necrosis with 0.05 and 0.5 mg/kg of d,l-propranolol (a level at which direct “membrane stabilizing” effects are insignificant) suggested that direct effects were not essential for protection. The negative results with d-propranolol further support our conclusion that propranolol reduces myocardial ischemic injury through beta adrenergic blockade
From the Department of Pathology, Northwestern University Medical School, Chicago, III. This study was supported in part by Grant HE 08729 and contract 72-2984 from the National Heart and Lung Institute, National Institutes of Health, Bethesda, Md. and a grant from the Chicago and Illinois Heart Association, Chicago, III. Predoctoral fellow (National Institutes of Health Grant GM 00131) during the time of study. Address for reprints: Keith Reimer, MD, Duke University Medical Center, Department of Pathology, Durham, N. C. 27710. l
520
March 31, 1976
rather than through direct myocardial
actions.
Our work with the effect of propranolol on acute myocardial infarction began in 1965, when we assessed whether the drug could prevent the ventricular fibrillation that often occurs in dogs at the onset of reperfusion after temporary coronary occlusion. We found that propranolol had no effect on reflow ventricular fibrillation but observed incidentally that it virtually eliminated the small subendocardial foci of myocardial necrosis associated with a 20 minute transient episode of ischemia.l Subsequently, we tested the potential protective effect of propranolol on ischemic cells in much greater detail.‘e4 Using histologic techniques to measure the amount of necrotic tissue, we found that propranolol reduced the amount of necrosis that develops in the dog heart after 40 minutes of temporary coronary occlusion or 24 hours of permanent coronary ligation. Occlusion of the circumflex branch of the left coronary artery results in transmural ischemia of the posterolateral wall of the heart but is not followed by cell death in all areas of the ischemic myocardium; the first cells to die are those in the subendocardium. In untreated dogs, 40 minutes of temporary occlusion of the left circumflex coronary artery produces necrosis (cell death) in about half the cells of the posterior papillary muscle (Fig. I), and the necrotic area extends laterally throughout the subendocardial myocardium supplied by this vessel. Tracer microsphere studies have demonstrated that these initial areas of cell death are areas of severe ischemia, that is, regions in which the collateral arterial flow is less than 15 percent of
The American Journal of CARDIOLOGY
Volume 37
PROPRANOLOL DURING CORONARY OCCLUSION-REIMER
the flow found before occlusion or in adjacent nonischemic myocardium.4 Propranolol therapy reduces the amount of necrosis that develops in areas of severe ischemia after a 40 minute temporary occlusion2 and thus prevents cell death in these regions. When ischemia is prolonged to 3 hours in untreated dogs, cell death extends into the midmyocardium and by 24 hours often is transmural (Fig. 1). Continuous propranolol treatment in dogs with 24 hour coronary ligation does not prevent necrosis in the subendocardial region of severe ischemia but does prevent cell death in much of the subepicardial myocardium and thereby produces a significant reduction of infarct size.3 Together, the results of these studies indicate that propranolol can delay the development of myocardial cell death in areas of severe ischemia and can prevent necrosis from developing in areas of moderate or mild ischemia.5 This study was undertaken to gain insight into the mechanism of the protective action of propranolol. The initial experiments cited were performed with a large dose of d,l-propranolol (5 mg/kg) that showed both beta adrenergic blocking and direct membrane stabilizing effects. The direct effects occur with both d- and 1-propranolol, but 1-propranolol is at least 50 times more potent than d-propranolol in inducing beta a!drenergic blockade. Our first purpose was to determine whether protection against necrosis was related to beta adrenergic blockade or to direct effects alone. This was accomplished in the 40 minute temporary occlusion model by (1) establishing the dose/response relation for cardiac protection in comparison with the dose/response relation for beta blockade, and (2) by comparing infarct size in dogs treated with d-propranolol and with the racemic mixture. A second purpose of this study was to determine whether propranolol could salvage myocardium when given after ischemic injury. We therefore gave one group of dogs propranolol 30 minutes after occlusion (10 minutes before release of a 40 minute occlusion) and compared the subsequent necrosis in this group with that in untreated and pretreated groups.
ET AL.
the heart removed for gross and histologic analysis. Data from dogs that did not show S-T segment elevation after occlusion and dogs that died suddenly or manifested thrombi at the site of occlusion were rejected from quantitative analysis. The left ventricle of each heart was opened, and the endocardial surface of the heart and longitudinal sections of the posterior papillary muscle were photographed for gross assessment of the extent of necrosis. Histologic sections were cut from four serial slices of the posterior papillary muscle. Sections from each slice were stained with hematoxylin-eosin, periodic acid-Schiff reaction for glycogen, and Heidenhain’s variant of Mallory’s connective-tissue method. Areas of necrosis were identified on sections stained with hematoxylin-eosin. Quantitation of the area of involvement was performed using either the periodic acidSchiff or connective tissue stain because the low power contrast between necrotic and normal areas was much better with these stains. Either the periodic acid-Schiff- or the connective-tissue-stained section from each slice of posterior papillary muscle was placed in a photographic enlarger,
Material and Methods Experimental design: Details of the experimental design have been reported.2 Healthy mongrel dogs of either sex, 9 to 18 kg, were fasted overnight and anesthetized with intravenously administered sodium pentobarbital, 30 mg/kg body weight. Each dog was intubated and ventilated with room air at a rate of 300 ml/kg per min. Lead II of the electrocardiogram and the femoral blood pressure (through a saphenous arterial catheter) were monitored continuously on a Grass model 5 polygraph. The heart was exposed through a left thoracotomy and pericardiotomy. A segment of the circumflex branch of the left coronary artery was isolated 1 to 2 cm from the aorta. The segment employed was always distal to the left atria1 branch and proximal to the first large ventricular branch. In all groups, the artery was nontraumatically occluded for 40 minutes and then the occlusion was released to restore flow. Dogs that survived the temporary ischemic injury were killed 2 to 4 days later and
FIGURE 1. Gross photographs of transmural longitudinal sections (endocardium upper, chordae tendineae to left) through the posterior papillary muscles of three representative dogs subjected to (from top to bottom) 40 minutes, 3 hours and 24 hours of occlusion of the left circumflex coronary artery. In the 40 minute and 3 hour studies temporary occlusion was followed by reflow; all three dogs were killed at 24 hours. Forty minutes of ischemia (top) results in necrosis (light areas) in the subendocardial myocardium (including posterior papillary muscle), but the other half of the wall is reversibly injured and survives. With a 3 hour occlusion (middle), the necrosis extends deeper into the midmyocardium and there is hemorrhage in the papillary muscle (indicating vascular injury). With 24 hours of Neischemia (bottom) the infarct frequently becomes transmural. crosis extends beyond a midmyocardlal zone of hyperemia and only small foci of subepicardial muscle are spared.
March 31, 1976
The American Journal of CARDIOLOGY
Volume 37
521
PROPRANOLOL
DURING
CORONARY
OCCLUSION-REIMER
ET
AL.
I
TABLE
Experimental
Study
Group Dogs Accepted for Analysis
Short-Term Dogs Group Untreated
Survivors
of
Necrosis
(no.)
(no.)
20
11
8
20
9
6
11
7
(no.)
d,l-propranolol’ (preoccluslon) 5 mgikg 0.5 mg/kg 0.05
mglkg
0.005
mgtkg
18
10
:
19
8
8
20
9
6
d,l-propranolol 130
minutes
after
occlusion) 5 w/kg d-propranolol (preocclusion) 5 mglkg
7
2
2
2.5
8
4
4
mg/kg
and the image printed on photographic paper or traced on heavyweight paper. The necrotic and surviving areas of each photograph or tracing were cut out and weighed, and the percent necrosis was calculated. By summing the areas from the three or four slices through the posterior papillary muscle, one obtained an estimate of the volume proportion of the muscle that was necrotic. Since in 40 minute temporary studies necrosis is largely confined to the subendocardial zones and particularly to the posterior papillary muscle, percent necrosis was calculated from a reference base including the longitudinal fibers of the posterior papillary muscle (anatomic posterior papillary muscle).
TABLE
Protocol: Eight experimental groups were studied. The number of dogs meeting the criteria cited for analysis in each group is shown in Table I. d,l-propranolol or d-propranolol in the doses shown was given as a bolus injection through the femoral arterial catheter either 10 minutes before or 30 minutes after coronary occlusion (10 minutes before release). The dose-response curve for beta adrenergic blockade with d,l-propranolol was obtained in four sham-operated dogs. The heart rate response to cumulative doses of 0.01, 0.1 and 1 wg/kg of isoproterenol was plotted before and after administration of 0.005,0.05, 0.5 and 5 mg/kg cumulative doses of d,l-propranolol. In these four dogs the three doses of isoproterenol were given at 30 second intervals, and peak heart rate response to each was determined. Five minutes later, the first dose of propranolol was given and, after an additional 10 minute equilibration period, the isoproterenol sequence was repeated. This sequence was then repeated three times with increasing levels of d,l-propran0101. In addition, beta adrenergic blockade was assessed at the time of sacrifice in dogs with myocardial infarcts that had been treated with the smaller doses of d,l-propranolol or with d-propranolol. In these dogs the heart rate response to isoproterenol was plotted before and after repeating the same dose of d,l- or d-propranolol that had been used during coronary occlusion 24 hours earlier.
Results
Of the 123 dogs used in this study, 60 survived both coronary occlusion and release (Table I). The 63 dogs that died all had ventricular fibrillation, which was seen most frequently either during the first 10 minutes after occlusion or at the time of release. Of the 60 dogs that survived the immediate intervention, 12 were removed from the study on the basis of
II
Hemodynamic, Electrocardiographic and Histologic Dogs Treated with d,l- or d-Propranolol
Results of Coronary
Heart A Heart
Rate
20
After “0.
Group Untreated
Occlusion
in Untreated
Cardrac
Rate Perrpheral
Minutes
Effort
Index
After
Blood
Propranolol
Occlusion
20
(beats/min)
(beatsimin)
After
Occlusion
123193
+ 7.116.6
17.1
+ 1.3
0.54
? 0.07
8
7
Pressure
Dogs and
Minutes
([HR
XSBPI 20
)/IO00
Minutes
After
Peak S-T
Occlusion
Elevation
Percent Necrosis
(mv)
51
* 8
139
i
123
+ 12
116184
I
16.1114.6
14.6
i
3.0
0.23
? 0.02
135’
9
126196
+ 13.8/13.1
17.4
t 2.8
0.32
* 0.06
21
+ 4+
27
i
d,l-propranolol (preocclusion) 5 mg/kg
6
-19
0.5
6
-15i-5”
8
-1
mg/kg
0.05
mglkg
0.005
8
mglkg
d,l-propranolol (30 mrnutes
after
occlusron)
+4
6
-2
2 4
-10 -3
i
6$
8
16i9+
+ 10
120
i
4+
105184
f 8.617.6
12.8
* 1 .4
0.40
i
+ 2
146
i
10
1431106
? 3.213.8
20.8
? 1 .5
0.55
* 0.11
32?
i
148
i
17
1401103
? 6.414.7
20.8
? 2.4
0.53
+ 0.11
31
?9
+ 8.719.7
10.8 18.8
t 2.2
0.60 0.50
? 0.09
45 40
* 7
2.1
0.07
7+ 11
d-propranolol (preocclusron) 5 mg/kg 2.5
mqiks
Values
represent
the
P (0.05
by
paired
P