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Protective effects on isolated hearts with developing infarction: Slow channel blockade by diltiazem versus beta-adrenoceptor antagonism by metoprolol
Protective Effects on Isolated Hearts With Developing Infarction: Slow Channel Blockade by Diltiazem Versus Beta-Adrenoceptor Antagonism by Metoprolol CHRISTIAN W. HAMM, MD, FRANCIS T. THANDROYEN, MRCP, and LIONEL H. OPIE, MD
Slow channel inhibitors (calcium antagonists) and beta-adrenoceptor antagonists may theoretically protect the infarcting myocardium by different mechanisms. Thus, beta-receptor antagonism reduces the oxygen demand by the combined effects of bradycardia, decreased arterial pressure, and reduced contractility, whereas slow channel inhibition chiefly increases coronary blood flow and decreases the afterload by peripheral vasodilation. The present study was designed to assess and compare the effects of the beta-receptor antagonist metoprolol with those of the calcium channel inhibitor diltiazem on cellular damage and mechanical function in the isolated working rat heart subject to coronary artery ligation. We compared the highest concentrations of both agents that could be tolerated in this preparation: diltiazem 4 X 10m7 M and metoprolol 10m4 M. These concentrations were equally effective in reducing enzyme release, which was taken as an index of cellular damage, and in preserving high energy phosphate contents in the in-
farcting myocardium. However, the 2 agents exhibited opposite effects on coronary flow rates and mechanical function. Compared with diltiazem metoprolol reduced total coronary flow, cardiac output, and left ventricular work and efficiency immediately after coronary artery ligation. However, diltiazem even favorably influenced the mechanical performance of the isolated heart compared with that of untreated control hearts during the 60 minutes of regional myocardial ischemia. These benefits were similarly apparent when the hearts were paced to the rate of control hearts. The presumed mechanism was coronary vasodilation, because afterload increased as left ventricular power production increased in this model. These results suggest that slow channel inhibition by diltiazem has advantages over beta-receptor antagonism by metoprolol in protecting the infarcting myocardium and that further evaluation of diltiazem in other preparations is warranted.
Modification of infarct size is currently an area of great endeavor in cardiac research.l Many pharmacologic and metabolic principles have been studied in the experimental animal with the aim of limiting the extent of cell necrosis caused by acute myocardial ischemia. One concept follows the hypothesis of Maroko et al.’ that the extent of ischemic injury is related to the myocardial oxygen balance. On this basis, several studies were un-
dertaken with beta-adrenoceptor antagonists and, more recently, slow channel inhibitors (calcium antagonists). Although both types of intervention could act directly on the heart to diminish oxygen requirements by reducing left ventricular contractile force and heart rate,2-4 in practice the depressive effect of calcium antagonists is thought to be modified by afterload reduction with a reflex increase in sympathetic tone, so that the sinus rate rarely decreases.” Furthermore, calcium antagonists can exert specific therapeutic effects in concentrations that do not cause myocardial depression.6 An additional effect of slow channel inhibitors is to further reduce oxygen demand and to increase oxygen supply to the ischemic myocardium by their peripheral and coronary artery vasodilating activity, which could result in reduced left ventricular afterload and
From the MRC-UCT lschaemic Heart Disease Research Laboratory, Department of Medicine, Groote Schuur Hospital and University of Cape Town, Cape Town, South Africa; and the Department of Cardiology, University Hospital, Hamburg, West Germany (Dr. Hamm). Manuscript received November 19, 1981; revised manuscript received March 2, 1982, accepted April 26, 1982. Address for reprints: Lionel H. Opie, MD, Medical School, Observatory 7925, South Africa.
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improved myocardial collateral flow, respectively.2 In contrast, there is some concern that beta-adrenoceptor antagonists, particularly noncardioselective compounds, might be associated with vasoconstrictive properties.7 Favorable results with experimental myocardial ischemia were reported for both beta-antagonists and calcium channel inhibitor+16 and extensive clinical trials are already projected. However, there is as yet no study that compares these two interventions directly under the same experimental condition. Therefore, the present investigation was designed to assess and compare the effects of beta-adrenoceptor antagonism and slow channel blockade on cellular damage and mechanical function during acute regional myocardial ischemia. For our study we chose the cardioselective beta-adrenoceptor antagonist metoprolol, reasoning that cardioselectivity should diminish the tendency of beta-antagonists to cause coronary vasoconstriction,7 and compared it with the recently introduced slow channel inhibitor diltiazem.12 Methods Perfusion
model: Male Long-Evans
rats (270 to 330 g) were anesthetized with ether and given 200 IU of heparin intravenously. The heart, weighing about 1 g (0.959 to 1.126 g, no significant differences between all groups), was rapidly excised and arrested in chilled perfusion fluid. After 15 minutes of retrograde preperfusion through the aorta, the working heart technique described by Neely et al.17 was started. In this model the isolated heart is filled by the left atrium at a constant filling pressure of 10 cm HsO and pumps spontaneously against a hydrostatic pressure load of 100 cm of water. Part of the fluid ejected from the left ventricle supplies the coronary arteries, drains into the right heart, and is collected from the pulmonary arteries as coronary flow. Aortic output at the top of the hydrostatic pressure column and coronary flow were measured by calibrated cylinders and added together for cardiac output. The perfusate used in this recirculating system was a Krebs-Henseleit bicarbonate buffer, maintained at 37°C and aerated with 95% 0s and 5% COs, and contained 11 mM of glucose as substrate. The oxygen uptake was determined from samples taken simultaneously from the left atria1 cannula and directly from the pulmonary artery measured as the oxygen tension (Radiometer, Copenhagen) and related to the coronary flow rate.i7 Acute regional myocardial ischemia was produced as de-
scribed by Kannengiesser et al.18 The left main coronary artery was ligated with 6-O silk by a 3/8 circle taper point needle (K-801 H, Ethicon). The stitch was placed with a small width about 2 mm below the aortic root. The coronary artery was ligated after 20 minutes of left atria1 perfusion and the heart was perfused an additional 60 minutes. Pharmacologically active agents were added to the perfusate 10 minutes before ligation. Measurements of coronary flow, cardiac output, peak aortic pressure, myocardial oxygen uptake, and release of lactate dehydrogenase were taken 10 minutes before and at the time of coronary artery ligation as well as 10,20,40, and 60 minutes thereafter. Enzyme release: The release of lactate dehydrogenase is a sensitive marker of myocardial cell viability ~oss,‘~ and the pattern of release from the isolated heart is similar to that of creatine kinase.20 We measured the arteriovenous differencezO of lactate dehydrogenase activity at the aforementioned time
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intervals with a Gilford 300-V spectrophotometer, using conventional assay techniques. The cumulative rate of enzyme loss was calculated, assuming linear functions, and expressed in international units per gram wet weight of heart. Hemodynamic measurements: Aortic pressure was measured by a Statham P23 Db pressure transducer and monitored on a Devices 2 channel M2 direct writer pen recorder. Total power production (work) of the heart was calculated as the sum of pressure and kinetic power21 according to the formulas: Pressure
power = p = 0.002222 X P, X CO
Kinetic power = k = l/(432 X 107) X [d(C0)3]/A2
X (T/Te)2
where P, = peak systolic pressure (mm Hg), CO = cardiac output (ml/min), d = density of perfusage (1 g/cm3), A = internal cross-sectional area of the aortic cannula (ems), T = cycle time (ms), and Te = ejection time (ms). The results are expressed in millijoules per second per gram heart weight. The efficiency of mechanical power production was calculated as total power divided by oxygen uptake (J/ml 02). Pharmacologically active agents: The beta-adrenoceptor antagonist metoprolol (Lopressore, Geigy)22 was used in a concentration of 1 X 10m4 M. The slow channel inhibitor diltiazem (Herbesser@, Tanabe Seiyaku)23 was used in a concentration of 4 X 10m7 M, which was the highest dose that did not cause complete heart block. The aforementioned concentrations chosen for each compound gave best protection against cellular damage in dose-response curves.24,25 Because both drugs significantly reduced the heart rate, a series of 6 hearts each was paced by the right atrium at the heart rate of control hearts (230 beats/min) in order to exclude heart rate effects on enzyme release and mechanical performance. Pacing was started immediately after administration of the drugs as soon as the heart rate dropped below 230 beats/min. Biochemical studies: To distinguish infarcting from noninfarcting myocardium, disulfine blue dye was injected into the left atrium just prior to freeze-clamping of unpaced hearts with Wollenberger tongs, precooled to the temperature of liquid nitrogen. Samples of the midischemic and uninvolved tissue of approximately 100 mg each were analyzed for adenosine triphosphate and phosphocreatine as previously described.2s If clear separation of stained and unstained tissue was not possible because of the timing or the angle of clamping, the hearts were discarded for biochemical analysis. Statistical procedures: Results were expressed as means f standard error of the mean. P values were calculated by the appropriate Student’s t test and p <0.05 was taken as significant, using 2-tailed values and corrections for unequal variances. If multiple comparisons were performed, Bonferroni’s correction was applied and the level of significance was raised accordingly.
Results of Metoprolol and Diltiazem on the Working Rat Heart Before Coronary Artery Ligation
Effects
The administration of metoprolol and diltiazem reduced the heart rate of the working heart from 244 f 8 to 168 f 14 beats/min (p
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TABLE
I
ET AL.
Effects of Metoprolol and Diltiarem on Coronary Flow, Oxygen Uptake, and Left Ventricular Power Production and Efficiency Before and After Coronary Artery Ligation Metoprolol
Ligation
Control (n = 10)
Nonpaced (n = 12)
Paced (n = 6)
Nonpaced (n = 10)
Diltiazem p vs Nonpaced Metoprolol
Paced (n = 6)
P vs Paced Metoprolol
Coronary flow Bimf’min) Pry rG!l, Oxygen uptake @l/min/g) BL Pre
I
;s Left ventricular power production (mJ/g/s) BL Pry ;:I Efficiency (J/ml 02) BL Pry ::I
15.0 15.0 7.6 8.1
158 163 93 87
f 0.7 f 0.7 f 0.2 rt: 0.2
13.2 11.8 6.2 6.6
f f f f
0.4 0.3 0.5+ 0.5+
155 136 72 75
f f f f
5 3’ 6+ 8
13.9 11.8 6.4 7.4
f f f f
0.5 0.3’ 0.31: 0.4
147 134 74 80
f f f f
4 4’ 2+ 3
f f f f-
5 4 5 3
15.49 15.41 7.09 4.91
f f f f
0.30 0.35 0.33 0.73
14.85 14.18 3.92 4.40
f f f f
0.56 0.44 0.66’ 0.63
13.97 13.27 5.28 5.71
5.94 5.76 4.63 3.46
f f f f
0.23 0.23 0.28 0.45
5.49 6.25 3.19 3.49
f f f f
0 31 0.17 0.34+ 0.31
5.67 5.92 4.23 4.27
14.2 f 0.6 15.8 f 0.6 8.6 f 0.8 87f07
164f 166 f 95 f 96 f
f 0.76 f 1.07 f 0.60+ f 0.45
f f f f
0.18 0.36 0.42 0.34
6 6 8 7
14.14 14.50 7.28 7.10
f f f f
0.44 0.53 0.51 0.35’
5.20 5.26 4.78 4.62
f f f f
0.16 0.14 0.10 0.30
<::01 <0.025 NS
<%Ol NS NS
13.7 16.9 9.3 9.8
* f f f
159 f 187 f 106 f 106 f
0.3 0.4 0.3’ 0.6
NS
5 8t 3 6:
<0:001
14.64 13.47 8.00 8.79
f f f f
0.77 0.77 0.55 0.65f
<::01
5.52 4.33 4.53 4.96
f f f f
0.17 0.17 0.23 0.20t
NS
NS
NS
* p0.025). Values are mean f standard error of the mean for number of hearts in brackets.
ml/min to 48.7 f 1.7 ml/min (p
on the Rat Heart
Coronary flow (Table I): Ligation of the left main coronary artery reduced the total coronary flow rate in untreated control hearts by 49% from 15.0 f 0.6 to 7.6 f 0.2 ml/min after 10 minutes. In the presence of metoprolol, the coronary flow decreased from 11.8 f 0.3 to
6.2 f 0.5 ml/min (47%) and with diltiazem from 15.8 f
0.6 to 8.6 f 0.8 ml/min (46%) (p CO.025 versus metoprolol). Hearts perfused with metoprolol generally had significantly lower flow rates than control hearts or hearts treated with diltiazem. It should be emphasized that these values of coronary flow refer to total flow, that is, the sum of ischemic and nonischemic zones. Mechanical function (Fig. 1): To assess mechanical function, we measured cardiac output and calculated left ventricular work. In control hearts cardiac output decreased 45% from 59.5 f 1.2 to 32.8 f 1.2 ml/min immediately after coronary ligation and progressively declined over the ensuing 60 minutes of acute regional myocardial ischemia. In hearts perfused with metoprolol cardiac output was initially reduced 64% to 17.5 f 2.5 ml/min (p CO.001 versus control), but was then maintained at that level, so that 60 minutes after ligation the metoprolol-treated and control hearts had similar cardiac outputs. With diltiazem, cardiac output first decreased similarly to control by 43% to 30.9 f 1.5 ml/min. However, these hearts showed no trend as did control hearts to go into cardiac failure, which in this model is defined as zero aortic output. Throughout the period of acute ischemia diltiazem maintained higher cardiac outputs than did metoprolol (p
increased cardiac output and left ventricular work in both drug-treated groups, yet the differences remained the same. However, pacing of diltiazem-treated hearts for 60 minutes after coronary artery ligation resulted
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Control o - -. -OMetoprolol . - - - -0 Metoprolol, paced w - Dihazem c -H Diltiazem, paced
* pco.02 * p < Q.QQl
-10
0
lo
20
30
Minutes after coronary
* p < 0,001 vs Control
1vsControl
40
50
Control
60
in significantly higher cardiac output and left ventricular work compared with control hearts (Table I). Efficiency of cardiac work (Table I): Enhanced mechanical function resulting from diltiazem could increase oxygen demand, which theoretically could be deleterious to the ischemic heart. Therefore, we measured oxygen uptake and related it to left ventricular work, thereby calculating the efficiency of cardiac work. Coronary artery ligation reduced the efficiency of work in control hearts from 5.76 f 0.23 to 4.63 f 0.28 J/ml 02 10 minutes after ligation and to 3.46 f 0.45 J/ml 0s 60 minutes after ligation. Metoprolol either left the efficiency unchanged or decreased it (10 minutes after ligation, unpaced hearts). Diltiazem was associated with
Minutes of ligation Figure 3. Release of lactate dehydrogenase over the time course of the experiments in coronary artery ligated (n = 10) and nonligated control hearts (n = 10) and hearts treated with metoprolol (n = 10) and diltiazem (n = 12).
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Metoprolol ---I
+
Diltlazem ---+
artery ligation
FIGURE 1. Effect of metoprolol 10m4 M(n = 10, paced n = 6) and diitiazem 4 X 10m7 A.4(n = 12, paced n = 6) on cardiac output. Diltiazem maintained higher cardiac output compared with metoprolol in unpaced (p
860
t--
FIGURE 2. Cumulative release of lactate dehydrogenase over 60 minutes after coronary ligation with metoprolol(1 X low4 M) and diltiazem (4 X lo-’ M) in paced and unpaced hearts. The 2 compounds were equally effective in reducing enzyme release.
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significantly higher efficiency values than metoprolol. However, this difference was not apparent if hearts were paced at equal heart rates. Enzyme release (Fig. 2): In control hearts, production of acute regional myocardial ischemia resulted in a release of lactate dehydrogenase of 8.88 f 0.60 U/h/g. This amount was reduced by about half by both metoprolol 1 X 10e4 A4 to 4.85 f 0.38 U/h/g (p
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TABLE II
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Noninfarcting
Phosphocreatine (wmol/g Fresh Weight)
lnfarcting
Noninfarcting
0 22
7 25 4 0.41
3.40 f 0 13
4.03 f 0 30
5 08 f
Preperfusion period, nonworking (n = 5) Control, working heart nonligated (n = 10)’ Control, ligated (n = 9) Metoprolol (n = 6) Diltiazem (n = 6) l
ET AL
Tissue High Energy Phosphate Contents in the lnfarcting and Noninfarcting Myocardium -___ _ Adenosine Triphosphate (hmol/g Fresh Weight)
Data + p
INFARCTION-HAMM
2.83 Jo 0.30 3.38 & 0 29 3.70 f 0 13+
0.60 f 0.05 1.08 f 0.17+ 0.97 f 0 12+
4.01 f 0 46 3.95 f 0.28 3.70 f 0.27
lnfarcting
1.21 f 0.11 1.72 f 0.23+ 1.46 f 0.14
from Kannengiesser et aLis 05. 01 versus control, ligated are mean f standard error of the mean.
zones of the heart in this model.18 However, even considerable admixture of ischemic and nonischemic tissue in the assay samples could not explain the more beneficial effect of diltiazem than metoprolol on adenosine triphosphate levels. Only metoprolol improved phosphocreatine values in the ischemic zone. Discussion Cellular damage: The present investigation demonstrated that the beta-adrenoceptor antagonist metoprolol and the slow channel inhibitor diltiazem exert substantial protection against cellular damage on acutely infarcting myocardium. Similar results have been obtained for other compounds in each drug group,8m1fi although not consistently.27-29 In the concentrations used, both interventions were equally effective in protecting myocardial cell integrity as assessed by enzyme loss in our preparation, and in addition preserved adenosine triphosphate in the infarcting tissue. Metoprolol, in addition, maintained phosphocreatine levels (Table II). However, the 2 agents exhibited opposing effects on the mechanical function of the heart after coronary ligation. Compared with diltiazem metoprolol caused deterioration of cardiac output and left ventricular work and efficiency immediately after production of acute myocardial ischemia. Effect on mechanical function: Some studies in larger animal9 have not revealed a depressant effect of beta-adrenoceptor antagonism on the mechanical function of the heart, but the compound used was practolol, which showed little cardiodepressant effect in some patient studies. A possible explanation for the relative absence of cardiodepressant effects by practolol is the intrinsic sympathomimetic activity that metoprolol does not possess. The concentration of metoprolol we used was probably higher than comparable doses of other beta-adrenoceptor antagonists used in most intact animal studies. In patients with acute infarction, intravenous metoprolol has also been used without precipitating left ventricular failure,31 but the patients initially had no clinical features of congestive heart failure, and therefore probably had relatively small infarcts. Ligation of the left main coronary artery in rats results in an infarct involving almost all of the
free wall of the left ventricles* and with a mean overall reduction of coronary flow to 57% of the preligation value.18 Therefore, the more extensive size of the ischemic zone in this preparation probably produced a more significant cardiac deterioration than that found in most patients with acute infarction. Improvement of the mechanical function of the heart mediated by diltiazem or other slow channel inhibitors has also been demonstrated in ischemic-reperfused and has been further confirmed in the preparations,33-s5 case of nifedipine by measurements of enhanced regional myocardial contractility in the ischemic myocardium of larger animals. 36 The effect on mechanical performance is considered to be related to the ability of these compounds to prevent deleterious intracellular calcium accumulation which can impair left ventricular contractile force and relaxation,37 but improved coronary perfusion to the ischemic zone is likely to be another important factor.ss,38 Verapamil, in contrast, appears to depress regional myocardial contractility in the ischemic but not the nonischemic myocardium, whereas propranoloi depresses both normal and ischemic myocardium.3g Preservation of cell integrity: A variety of mechanisms have already been proposed for the reduction of cellular damage in the acutely infarcting myocardium by beta-receptor antagonists and slow channel inhibitors.3,s,8J2 Our experiments show that protection can be achieved when heart rate is not reduced. Severity of cellular damage has been assessed by release of lactate dehydrogenase and the tissue content of high energy phosphates. It has to be noted that phosphocreatine, increased in the ischemic zone only by metoprolol, cannot be related to the extent of enzyme release40 in contrast to the level of adenosine triphosphate.41 It may be speculated that the elevated adenosine triphosphate content achieved by both metoprolol and diltiazem in ischemic myocardium could represent a specialized pool to which Bricknell and Opie*2 attributed a role in protecting the cell membrane. Myocardial perfusion: In addition to the beneficial effects of diltiazem on the calcium homeostasis in the ischemic myocardium, its vasodilating activity has to be considered as a mechanism of protection.saJs Dilti-
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LV work+
4 LV
INFARCTION-HAMM
work
j . .. tissue I’ ATP 4 +coronary
flow
+ LDH-rel:ase f
FIGURE 4. Comparative effects of metoprolol and diltiazem on acutely infarcting myocardium. Results are based on comparisons with control hearts in Tables I and Il. ATP = adenosine triphosphate; LDH = lactic dehydrogenase; LV = left ventricle
azem causes an increase in the blood flow in the peripheral zone of regional myocardial ischemia in the pig mode1.43T44 Because no evidence for a coronarv “steal” phenomenon43-45 with diltiazem has been reported (in contrast to nifedipine27), one may suppose from our experiments that there was a redistribution, which was not clearly detectable by measuring the total coronary flow rate. By this, oxygen supply to the ischemic tissue would be imnroved and tissue acidosis diminished through washout of lactate and hydrogen ions. Excess hydrogen ions not only interfere with key reactions of metabolic pathways, 46 but also represent another factor that has been associated with impairment of the contractile function.47. Even though metonrolol exhibited reduced total coronary flow-rates in our model, we cannot completely exclude a favorable redistribution of flow to the ischemic zone as found in the pig mode1.48 A reduced washout of enzymes is possible in the case of metoprolol, so that enzyme release was falsely low; this sequence is rendered unlikely by increased values for high energy phosphates in the infarcting tissue. In addition, no facilitated washout is apparent for diltiazem (Fig. 2). Therefore, equal protection of cell integrity in the presence of significantly different flow rates suggests that mechanisms other than nerfusion effects are involved in mediating preservation of ischemic myocardium. However, the opposing effects on mechanical function may possibly be related to the degree of myocardial perfusion. Critique of the model: The isolated working rat heart with coronary artery ligation is a model with both advantages and disadvantages. The dynamic function of the heart can be studied under reproducible and controllable conditions.18 whereas the severitv of cellular damage induced by coronary artery ligation can be measured continuously and accurately by enzyme release.20 A limitation of this preparation is that it fails to allow physiologic interaction of the heart with the peripheral circulation. The preparation is severed from the autonomic nervous system, quite unlike the situation in patients with infarction. The results obtained with this model cannot directly be extrapolated to the human heart, because heart rate, cardiac output, and coronary flow rates of the isolated rat heart are not comparable to those in man. The situation in any animal .z
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model when healthy coronary arteries are abruptly ligated may not be relevant to the much more complex coronary artery anatomy in patients when occlusive disease can antedate acute infarction by many years. Another outstanding question is whether the beneficial effects of diltiazem administration before coronary artery ligation could also be achieved after the onset of the infarction process. However, most of the interventions initially applied before coronary artery occlusion1 have also been effective within the first hours after coronary occlusion.:~ The experimental concentrations of the 2 potentially cardioprotective agents are also open to criticism. With diltiazem, therapeutic concentrations in patients may reach 3 X lop7 g/ml or 7.3 X low7 M.5 Similar concentrations inhibit smooth muscle excitation coupling in the coronary arteries of the pig2 and increase spontaneous cycle length in the isolated sinoatrial node in the rabbit.” Hence, the concentration (4 x lop7 M) used in this study may well be of therapeutic importance in clinical practice. With metoprolol, a relatively large dose (10e4 M) was used that is well above the effective blood level of about 10e7 M.4g At such high concentrations, cardioselectivity might well have been lost, hence tending to reduce coronary flow by an unopposed alpha-mediated vasoconstriction. However, because our study was based on a comparison of 2 similarly potent protective concentrations, 10B4 M of metoprolol was required to be as effective as 4 X 10e7 M of diltiazem.23,24 This concentration of metoprolol was also required to antagonize isoproterenol lop6 M. The local concentration of norepinephrine in ischemic myocardium is likely to be about 10h6 M.40 At such very high concentrations, effects other than beta-antagonism, such as membrane protection, are likely to be relevant. Such comparisons apply strictly to the isolated heart. In patients with acute myocardial infarction, circulating catecholamine concentrations (largely norepinephrine) are increased to about 5 X low9 Mso; such concentrations of norepinephrine fail to increase enzyme release from the isolated rat heart.40 Hence, our data may possibly have application in acute myocardial infarction even in the absence of added catecholamine stimulation. Conclusion: The present study demonstrates that beta-adrenoceptor antagonism by metoprolol at large doses and slow channel inhibition by diltiazem at apparently therapeutic doses are equally effective in reducing cellular damage in the acutely infarcting myocardium but that there are opposing effects on the mechanical function of the heart. Diltiazem offered substantial cardioprotection in the presence of significantly better mechanical performance than metoprolol. Whereas metoprolol reduced cardiac output and left ventricular work compared with untreated control hearts, these variables appear to be influenced in a favorable way by diltiazem. Although our results were obtained in an isolated, perfused rat heart model, the principles involved could be of considerable importance in a clinical setting. Patients with acute myocardial infarction often have restricted ventricular function, Volume 50
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especially when the infarct is large, as in our model.“’ Further myocardial depression by beta-receptor antagonism could initiate cardiac decompensation and result in cardiogenic shock, whereas our studies raise the expectation that diltiazem could improve the mechanical function possibly by facilitating blood flow to the ischemic zone. Although conclusions drawn from diltiazem may not apply to other slow channel inhibitors and evidence indicates that further experimental data in other models are required, both theoretical and experimental data suggest that slow channel blockade could become a more promising therapeutic approach than beta-adrenoceptor antagonism in minimizing myocardial infarct size
(Fig. 4). Acknowledgment Dr. C. Hamm thanks the Chris Barnard Fund and the Deutsche Forschungsgemeinschaft for generous support. This work forms part of an ongoing program of the Medical Research Council of South Africa, who are also thanked for support. We thank Professor W. Bleifeld for cooperation and advice. References 1. Maroko PR. Kjekshus JK, Sobel BE, et al. Factors influencing infarct size following experimental coronary artery occlusions. Circulation 1971;43: 67-82. 2. Fleckensteln A. Specific oharmacoloov of calcium in mvocardium. cardiac pacemakers, and.vascul& smooth n%scle. Ann Rev bharmacof Toxicol 1977:17:149-166. 3. Kloner RA, Braunwaid E. Observations on experimental myocardial ischemia. Cardiovasc Res 1980;14:371-395. 4. Opie LH, Thomas M. Propranolol and experimental myocardial infarctjon substrate effects. Postgrad Med J 1976;52(Suppl 4): 124-132. 5. Kawai C, Konishi T, Matsuyama E. Dkazaki H. Comparative effects of three calcium antagonists, diltiazem, verapamil and nifedipine on the sinoatrial and atrioventricular nodes. Circulation 1981:63:1035-1042. 6. Feriinz J, Easthope JL, Aronow WS. Effects of verapamil on myocardial performance in coronary disease Circulation 1978;12:28-33. 7. Marshall RJ, Parr&i JR. Comparative effects of propranolol and practolol in the early stages of experimental canine myocardial infarction. t3r J Pharmacol 1976;57:295-303. 9. Henry PD. Hucaieib R, Borda LJ, Roberts R, WIlliamson JR, Sobei BE. Effects of nifedipine on myocardial and ischemic injury in dogs Circ Res 1978;43:372-380. 9. Nagao T, Matlib MA, Frankiln D, Millard RW, Schwartz A. Effects of diltiazem, a calcium antagonist, on regional myocardial function and mitochondria after brief coronary occlusion. J Mol Cell Cardiol 1980;12:2943. IO. Relmer KA, Lowe JE, Jennings RE. Effect of the calcium antagonlst verapamil on necrosis following temporary coronary artery occlusion In dogs. Circulation 1977;55:581-587 11. Smith HJ, Singh BN, Nlsbet HD, Norris RM. Effects of verapamil on infarct size following experimental coronary occlusion. Cardiovasc Res 1975;9. 569-578. 12. Weishaar R, Ashikawa K, Bing RJ. Effect of diltiazem. a calcium antagonist, on myocardial ischemia. Am J Cardiol 1979:43:1137-l 143. 13. Wende W, Bieffeid W, Meyer J, Stuhiin HW. Reducbon of the size of acute experimental myocardial Infarction by verapamil. Basic Res Cardiol 1975;70:198-208. 14. Libby P, MarokoPR, Coveil JW, Mailoch Cl, Ross J Jr, Braunwaid E. Effect of practolol on the extent of myocardial ischemic in)uty after experimental coronary occlusion -. and its effects on ventricular function in the normal and lscnemlc neart. tiardiovasc Res 1973;7.167-173. 15. Miura M, Thomas R, Ganz W, et al. The effect of delay in propranolol administration on reduction of myocardial infarct size after experimental coronary artery occlusion in dogs Circulation 1979;59:1148-1157. 16. Reimer KA, Rasmussen MM, Jennings RB. Reduction by propranolol of my-dial necrosis following temporary coronary artery occlusion In dogs. Circ Res 1973;33:353-363 17. Neely JR, Liebermeiater H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart Am J Physiol 1967;212:804-814. 18. KannangiasserGJ, Lubbe WF, Opie LH. Experimental myocardial infarction with left ventricular failure in the isolated perfused rat heart Effects of lsoproterenol and pacing. J Mol Cell Cardiol 1975;7:135-151. 19. Van der Laarse A, Holiaar L, Van der Vatk LJM. Release of alpha hydfoxybutyrate from neonatal rat heart cell cultures exoosed to anoxia and reoxygenation. Comparison with impairment of strudture and function of damaged cardiac cells. Cardiovasc Res 1979;13.345-353.
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20. De Leiris J, Opie LH. Effect of substrates and of coronary artery ligation on mechanlcal performance and on release of lactate dehydrogenase and creatlne phosphoklnase in isolated working rat hearts. Cardiovasc Res 1978.12:585-596 21. Kannengiesser GJ, Opie LH, van der Werff TJ. Impaired cardiac work and oxygen uptake after reperfusion of regionally lschemic myocardlum J Mol Cell Cardiol 1979,i 1’197-207 22. Frishman W, Silverman R. Cltmcal pharmacology of the new beta-adrenerglc blocking drugs Part 2 Physiologic and metabotic effects Am Heart J 1979,97:797-807 23. Saikawa T, Nagamoto Y, Arita M. Electrophysiologtc effects of diltiazem. a slow channel inhibltor, on camne cardiac fibers Jpn Heart J 1977;18: 235-245 24. Hamm CW, Opie LH. ProtectIon of lnfarctlng card&urn by slow channel inhfbrtors (abstr) Am J Cardiol 1982,49-942 25. Hamm CW, Opie LH. Erhaltung akut lschamlschen Myokards durch Diltiazem Amsterdam Excerpta Medica, In press 26. Opie LH, Mansford KRL, Owen P. Effects of Increased heart work on glycolysis and adenlne nucleotldes in the perfused heart of normal and diabetic rats Biochem J 197 1,124.475-490 27. Peter T, Heng MK, Singh BN, et al. Failure of htgh doses of propranolol to reduce experimental myocardlal lschemic damage Circulation 1978:57: 534-540 28. Karlsberg RP, Henry PD. Ahmed SA, Sobei BE, Roberts R. Lack of protectlon of ischemic myocardtum by verapamil in conscious dogs Europ J Pharmacol 1977,42 339-346 29. Selwyn AP, Welman E, Fox K, Horlock P, Pratt T, Keiin M. The effects of nifedlplne on acute experimental myocardial lschemla and infarction in dogs. Circ Res 1979;44 16-23. 30. Libby P, Maroko PR, Covell JW, Malloch Cl, Ross J Jr, Braunwald E. Effect of practolol on the extent of myocardial ischemic injury after experimental coronary occlusion and its effects on ventricular function in the normal and lschemic heart Cardiovasc Res 1973,7.167-173 31. Waagstein F, Hjalmarson A. Double-blind study of the effect of cardloselective beta-blockade on chest paln in acute myocardlal infarction Acta Med Stand 1976,587(Suppl).201-208. 32. Fishbein MC, Hare CA, Gissen SA, Spadaro J, Maclean 0, Maroko P. Identification and quantification of histochemical border zones during evc$tlon of myocardial infarctlon in the rat Cardlovasc Res 1980;14:4133. Jolly SR, Menahan LA, Gross GJ. Diltiazem in myocardlal recovery from global tschemia and reperfuslon. J Mol Cell Cardlol 1981;13:359-372. 34. Weishaar RE, Bing RJ. The beneficial effect of a calcium channel blocker, ddtiazem. on the ischemlc reperfused heart J Mol Cell Cardiol 1980;12: 993-1009 35. Sherman LG, Liang CS, Boden WE, Hood WB. The effect of verapamil on mechanical performance of acutety ischemic and reperfused myocardium In the conscious dog Circ Res 1981;48:224-232. 36. Henry PD, Shuchleib R, Clark RE, Perez JE. Effect of nifedipine on myocardlal ischemla analysis of collateral flow, oulsatile beat and reaional muscle shortening Ati J Cardiol 1979;44.817’-824 37. Henry PD, Shuchleib R, Davis J, Weiss ES, Sobel BE. Myocardial contractufe and accumulation of mltochondrlal catclum in lschemic rabbit heart Am J Physrol 1977;233:H677-684. 38. Jolly SR, Gross GJ. Improvement of lschemlc myocardlal blood flow following a new calcium antagonist Am J Physiol 1980;239,Hl63-HI71 39. Smith HJ, Goldstein RA, Grlffith JM, Kent KM, Epstein SE. Regional contractiltty Selective depression of lschemtc myocardium by verapamil. Circulation 1976.54:629-635. 40. Waldenstrom Ak, Hjalmarson AC, Thorneli L. A possible rote of noradrenaline In the development of myocardial Infarction Am Heart J 1978.95:43-5 1, 41. Gebhard MM, Denkhaus H, Sakai K, Spleckermann PG. Energy metabolism and enzvme release J Mol Med 1977.2 271-783 -_-. 42. Brickneil OL, Opie LH: Effectsof &b&aies on tissue metabohc changes in the Isolated rat heart during underperfusion and on release of lactate dehydrogenase and arrhythmias during reperfusion Circ Res 1978;43: 102-115 43. Millard RW. Changes in cardiac mechanics and coronary blood flow of regtonally lschemic porcine myocardium Induced by diltiazem Chest 1980:78(Suppl 1):193-199 44. Franklin D, Millard RW. Nagao T. Responses of coronary collateral flow and dependent myocardial mechanical function to the calcium antagonist, diltiazem Chest 1980;78(Suppl 1),200-204 45. Nagao T, Murata S, Sat0 M. Effects of diltiazem (CRD-401) on developed coronary collaterals in the dog Jpn J Pharmacot 1975;25:281-288, 46. Opie LH. Metabolic regulation In ischemia and hypoxia. Effects of regional tschemla on metabolism of glucose and fatty acids Circ Res 1976;38(Suppl 1) l-52-l-74. 47. Williamson JR, Schaffer SW, Ford C, Safer B. The cellular basis of ischemia and Infarction. Contribution of tissue acidosis to ischemic injury in the perfused rat heart Circulation 1976;53jSuppl 1):1-3-i-14. 48. Muller CA, Hamm CW, Opie LH, Pelsach M, Glhwala D. Effect of betaadrenoceptor antagonism on regional left ventricular blood flow and incidence of ventflcular fibrillation after coronary artery ligation in pigs (abstr). Proc 8th Int Cong Pharmacol. Tokyo, 1981 49. Regardh CG, Johnsaon G, Jordo L, Lundborg P, Persson BA, Ronn 0. Plasma concentrations and beta-blocking effects in normal volunteers after tntravenous doses of metopfolol and propranotol J Cardiovasc Pharmacol 1980;2:715-723. 50. Vetier NJ, StrangeRC, Adams W, Oliver MF. lnitlat metabolic and hormonal response to acute myocardial infarction Lancet 1974;1:284-289. 51. Bleifeld W, Mathey D, Hanrath P, Buss H, Effert S. Infarct size estimated from serial serum creatine phosphokinase in relation to left ventricular hemodynamics Circulation 1977.55:303-311
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Slow channel inhibitors (calcium antagonists) and beta-adrenoceptor antagonists may theoretically protect the infarcting myocardium by different mechanisms. Thus, beta-receptor antagonism reduces the oxygen demand by the combined effects of bradycardia, decreased arterial pressure, and reduced contractility, whereas slow channel inhibition chiefly increases coronary blood flow and decreases the afterload by peripheral vasodilation. The present study was designed to assess and compare the effects of the beta-receptor antagonist metoprolol with those of the calcium channel inhibitor diltiazem on cellular damage and mechanical function in the isolated working rat heart subject to coronary artery ligation. We compared the highest concentrations of both agents that could be tolerated in this preparation: diltiazem 4 X 10m7 M and metoprolol 10m4 M. These concentrations were equally effective in reducing enzyme release, which was taken as an index of cellular damage, and in preserving high energy phosphate contents in the in-
farcting myocardium. However, the 2 agents exhibited opposite effects on coronary flow rates and mechanical function. Compared with diltiazem metoprolol reduced total coronary flow, cardiac output, and left ventricular work and efficiency immediately after coronary artery ligation. However, diltiazem even favorably influenced the mechanical performance of the isolated heart compared with that of untreated control hearts during the 60 minutes of regional myocardial ischemia. These benefits were similarly apparent when the hearts were paced to the rate of control hearts. The presumed mechanism was coronary vasodilation, because afterload increased as left ventricular power production increased in this model. These results suggest that slow channel inhibition by diltiazem has advantages over beta-receptor antagonism by metoprolol in protecting the infarcting myocardium and that further evaluation of diltiazem in other preparations is warranted.
Modification of infarct size is currently an area of great endeavor in cardiac research.l Many pharmacologic and metabolic principles have been studied in the experimental animal with the aim of limiting the extent of cell necrosis caused by acute myocardial ischemia. One concept follows the hypothesis of Maroko et al.’ that the extent of ischemic injury is related to the myocardial oxygen balance. On this basis, several studies were un-
dertaken with beta-adrenoceptor antagonists and, more recently, slow channel inhibitors (calcium antagonists). Although both types of intervention could act directly on the heart to diminish oxygen requirements by reducing left ventricular contractile force and heart rate,2-4 in practice the depressive effect of calcium antagonists is thought to be modified by afterload reduction with a reflex increase in sympathetic tone, so that the sinus rate rarely decreases.” Furthermore, calcium antagonists can exert specific therapeutic effects in concentrations that do not cause myocardial depression.6 An additional effect of slow channel inhibitors is to further reduce oxygen demand and to increase oxygen supply to the ischemic myocardium by their peripheral and coronary artery vasodilating activity, which could result in reduced left ventricular afterload and
From the MRC-UCT lschaemic Heart Disease Research Laboratory, Department of Medicine, Groote Schuur Hospital and University of Cape Town, Cape Town, South Africa; and the Department of Cardiology, University Hospital, Hamburg, West Germany (Dr. Hamm). Manuscript received November 19, 1981; revised manuscript received March 2, 1982, accepted April 26, 1982. Address for reprints: Lionel H. Opie, MD, Medical School, Observatory 7925, South Africa.
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improved myocardial collateral flow, respectively.2 In contrast, there is some concern that beta-adrenoceptor antagonists, particularly noncardioselective compounds, might be associated with vasoconstrictive properties.7 Favorable results with experimental myocardial ischemia were reported for both beta-antagonists and calcium channel inhibitor+16 and extensive clinical trials are already projected. However, there is as yet no study that compares these two interventions directly under the same experimental condition. Therefore, the present investigation was designed to assess and compare the effects of beta-adrenoceptor antagonism and slow channel blockade on cellular damage and mechanical function during acute regional myocardial ischemia. For our study we chose the cardioselective beta-adrenoceptor antagonist metoprolol, reasoning that cardioselectivity should diminish the tendency of beta-antagonists to cause coronary vasoconstriction,7 and compared it with the recently introduced slow channel inhibitor diltiazem.12 Methods Perfusion
model: Male Long-Evans
rats (270 to 330 g) were anesthetized with ether and given 200 IU of heparin intravenously. The heart, weighing about 1 g (0.959 to 1.126 g, no significant differences between all groups), was rapidly excised and arrested in chilled perfusion fluid. After 15 minutes of retrograde preperfusion through the aorta, the working heart technique described by Neely et al.17 was started. In this model the isolated heart is filled by the left atrium at a constant filling pressure of 10 cm HsO and pumps spontaneously against a hydrostatic pressure load of 100 cm of water. Part of the fluid ejected from the left ventricle supplies the coronary arteries, drains into the right heart, and is collected from the pulmonary arteries as coronary flow. Aortic output at the top of the hydrostatic pressure column and coronary flow were measured by calibrated cylinders and added together for cardiac output. The perfusate used in this recirculating system was a Krebs-Henseleit bicarbonate buffer, maintained at 37°C and aerated with 95% 0s and 5% COs, and contained 11 mM of glucose as substrate. The oxygen uptake was determined from samples taken simultaneously from the left atria1 cannula and directly from the pulmonary artery measured as the oxygen tension (Radiometer, Copenhagen) and related to the coronary flow rate.i7 Acute regional myocardial ischemia was produced as de-
scribed by Kannengiesser et al.18 The left main coronary artery was ligated with 6-O silk by a 3/8 circle taper point needle (K-801 H, Ethicon). The stitch was placed with a small width about 2 mm below the aortic root. The coronary artery was ligated after 20 minutes of left atria1 perfusion and the heart was perfused an additional 60 minutes. Pharmacologically active agents were added to the perfusate 10 minutes before ligation. Measurements of coronary flow, cardiac output, peak aortic pressure, myocardial oxygen uptake, and release of lactate dehydrogenase were taken 10 minutes before and at the time of coronary artery ligation as well as 10,20,40, and 60 minutes thereafter. Enzyme release: The release of lactate dehydrogenase is a sensitive marker of myocardial cell viability ~oss,‘~ and the pattern of release from the isolated heart is similar to that of creatine kinase.20 We measured the arteriovenous differencezO of lactate dehydrogenase activity at the aforementioned time
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intervals with a Gilford 300-V spectrophotometer, using conventional assay techniques. The cumulative rate of enzyme loss was calculated, assuming linear functions, and expressed in international units per gram wet weight of heart. Hemodynamic measurements: Aortic pressure was measured by a Statham P23 Db pressure transducer and monitored on a Devices 2 channel M2 direct writer pen recorder. Total power production (work) of the heart was calculated as the sum of pressure and kinetic power21 according to the formulas: Pressure
power = p = 0.002222 X P, X CO
Kinetic power = k = l/(432 X 107) X [d(C0)3]/A2
X (T/Te)2
where P, = peak systolic pressure (mm Hg), CO = cardiac output (ml/min), d = density of perfusage (1 g/cm3), A = internal cross-sectional area of the aortic cannula (ems), T = cycle time (ms), and Te = ejection time (ms). The results are expressed in millijoules per second per gram heart weight. The efficiency of mechanical power production was calculated as total power divided by oxygen uptake (J/ml 02). Pharmacologically active agents: The beta-adrenoceptor antagonist metoprolol (Lopressore, Geigy)22 was used in a concentration of 1 X 10m4 M. The slow channel inhibitor diltiazem (Herbesser@, Tanabe Seiyaku)23 was used in a concentration of 4 X 10m7 M, which was the highest dose that did not cause complete heart block. The aforementioned concentrations chosen for each compound gave best protection against cellular damage in dose-response curves.24,25 Because both drugs significantly reduced the heart rate, a series of 6 hearts each was paced by the right atrium at the heart rate of control hearts (230 beats/min) in order to exclude heart rate effects on enzyme release and mechanical performance. Pacing was started immediately after administration of the drugs as soon as the heart rate dropped below 230 beats/min. Biochemical studies: To distinguish infarcting from noninfarcting myocardium, disulfine blue dye was injected into the left atrium just prior to freeze-clamping of unpaced hearts with Wollenberger tongs, precooled to the temperature of liquid nitrogen. Samples of the midischemic and uninvolved tissue of approximately 100 mg each were analyzed for adenosine triphosphate and phosphocreatine as previously described.2s If clear separation of stained and unstained tissue was not possible because of the timing or the angle of clamping, the hearts were discarded for biochemical analysis. Statistical procedures: Results were expressed as means f standard error of the mean. P values were calculated by the appropriate Student’s t test and p <0.05 was taken as significant, using 2-tailed values and corrections for unequal variances. If multiple comparisons were performed, Bonferroni’s correction was applied and the level of significance was raised accordingly.
Results of Metoprolol and Diltiazem on the Working Rat Heart Before Coronary Artery Ligation
Effects
The administration of metoprolol and diltiazem reduced the heart rate of the working heart from 244 f 8 to 168 f 14 beats/min (p
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TABLE
I
ET AL.
Effects of Metoprolol and Diltiarem on Coronary Flow, Oxygen Uptake, and Left Ventricular Power Production and Efficiency Before and After Coronary Artery Ligation Metoprolol
Ligation
Control (n = 10)
Nonpaced (n = 12)
Paced (n = 6)
Nonpaced (n = 10)
Diltiazem p vs Nonpaced Metoprolol
Paced (n = 6)
P vs Paced Metoprolol
Coronary flow Bimf’min) Pry rG!l, Oxygen uptake @l/min/g) BL Pre
I
;s Left ventricular power production (mJ/g/s) BL Pry ;:I Efficiency (J/ml 02) BL Pry ::I
15.0 15.0 7.6 8.1
158 163 93 87
f 0.7 f 0.7 f 0.2 rt: 0.2
13.2 11.8 6.2 6.6
f f f f
0.4 0.3 0.5+ 0.5+
155 136 72 75
f f f f
5 3’ 6+ 8
13.9 11.8 6.4 7.4
f f f f
0.5 0.3’ 0.31: 0.4
147 134 74 80
f f f f
4 4’ 2+ 3
f f f f-
5 4 5 3
15.49 15.41 7.09 4.91
f f f f
0.30 0.35 0.33 0.73
14.85 14.18 3.92 4.40
f f f f
0.56 0.44 0.66’ 0.63
13.97 13.27 5.28 5.71
5.94 5.76 4.63 3.46
f f f f
0.23 0.23 0.28 0.45
5.49 6.25 3.19 3.49
f f f f
0 31 0.17 0.34+ 0.31
5.67 5.92 4.23 4.27
14.2 f 0.6 15.8 f 0.6 8.6 f 0.8 87f07
164f 166 f 95 f 96 f
f 0.76 f 1.07 f 0.60+ f 0.45
f f f f
0.18 0.36 0.42 0.34
6 6 8 7
14.14 14.50 7.28 7.10
f f f f
0.44 0.53 0.51 0.35’
5.20 5.26 4.78 4.62
f f f f
0.16 0.14 0.10 0.30
<::01 <0.025 NS
<%Ol NS NS
13.7 16.9 9.3 9.8
* f f f
159 f 187 f 106 f 106 f
0.3 0.4 0.3’ 0.6
NS
5 8t 3 6:
<0:001
14.64 13.47 8.00 8.79
f f f f
0.77 0.77 0.55 0.65f
<::01
5.52 4.33 4.53 4.96
f f f f
0.17 0.17 0.23 0.20t
NS
NS
NS
* p
ml/min to 48.7 f 1.7 ml/min (p
on the Rat Heart
Coronary flow (Table I): Ligation of the left main coronary artery reduced the total coronary flow rate in untreated control hearts by 49% from 15.0 f 0.6 to 7.6 f 0.2 ml/min after 10 minutes. In the presence of metoprolol, the coronary flow decreased from 11.8 f 0.3 to
6.2 f 0.5 ml/min (47%) and with diltiazem from 15.8 f
0.6 to 8.6 f 0.8 ml/min (46%) (p CO.025 versus metoprolol). Hearts perfused with metoprolol generally had significantly lower flow rates than control hearts or hearts treated with diltiazem. It should be emphasized that these values of coronary flow refer to total flow, that is, the sum of ischemic and nonischemic zones. Mechanical function (Fig. 1): To assess mechanical function, we measured cardiac output and calculated left ventricular work. In control hearts cardiac output decreased 45% from 59.5 f 1.2 to 32.8 f 1.2 ml/min immediately after coronary ligation and progressively declined over the ensuing 60 minutes of acute regional myocardial ischemia. In hearts perfused with metoprolol cardiac output was initially reduced 64% to 17.5 f 2.5 ml/min (p CO.001 versus control), but was then maintained at that level, so that 60 minutes after ligation the metoprolol-treated and control hearts had similar cardiac outputs. With diltiazem, cardiac output first decreased similarly to control by 43% to 30.9 f 1.5 ml/min. However, these hearts showed no trend as did control hearts to go into cardiac failure, which in this model is defined as zero aortic output. Throughout the period of acute ischemia diltiazem maintained higher cardiac outputs than did metoprolol (p
increased cardiac output and left ventricular work in both drug-treated groups, yet the differences remained the same. However, pacing of diltiazem-treated hearts for 60 minutes after coronary artery ligation resulted
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Control o - -. -OMetoprolol . - - - -0 Metoprolol, paced w - Dihazem c -H Diltiazem, paced
* pco.02 * p < Q.QQl
-10
0
lo
20
30
Minutes after coronary
* p < 0,001 vs Control
1vsControl
40
50
Control
60
in significantly higher cardiac output and left ventricular work compared with control hearts (Table I). Efficiency of cardiac work (Table I): Enhanced mechanical function resulting from diltiazem could increase oxygen demand, which theoretically could be deleterious to the ischemic heart. Therefore, we measured oxygen uptake and related it to left ventricular work, thereby calculating the efficiency of cardiac work. Coronary artery ligation reduced the efficiency of work in control hearts from 5.76 f 0.23 to 4.63 f 0.28 J/ml 02 10 minutes after ligation and to 3.46 f 0.45 J/ml 0s 60 minutes after ligation. Metoprolol either left the efficiency unchanged or decreased it (10 minutes after ligation, unpaced hearts). Diltiazem was associated with
Minutes of ligation Figure 3. Release of lactate dehydrogenase over the time course of the experiments in coronary artery ligated (n = 10) and nonligated control hearts (n = 10) and hearts treated with metoprolol (n = 10) and diltiazem (n = 12).
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Metoprolol ---I
+
Diltlazem ---+
artery ligation
FIGURE 1. Effect of metoprolol 10m4 M(n = 10, paced n = 6) and diitiazem 4 X 10m7 A.4(n = 12, paced n = 6) on cardiac output. Diltiazem maintained higher cardiac output compared with metoprolol in unpaced (p
860
t--
FIGURE 2. Cumulative release of lactate dehydrogenase over 60 minutes after coronary ligation with metoprolol(1 X low4 M) and diltiazem (4 X lo-’ M) in paced and unpaced hearts. The 2 compounds were equally effective in reducing enzyme release.
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significantly higher efficiency values than metoprolol. However, this difference was not apparent if hearts were paced at equal heart rates. Enzyme release (Fig. 2): In control hearts, production of acute regional myocardial ischemia resulted in a release of lactate dehydrogenase of 8.88 f 0.60 U/h/g. This amount was reduced by about half by both metoprolol 1 X 10e4 A4 to 4.85 f 0.38 U/h/g (p
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VERSUS METOPROLOL IN EXPERIMENTAL
Noninfarcting
Phosphocreatine (wmol/g Fresh Weight)
lnfarcting
Noninfarcting
0 22
7 25 4 0.41
3.40 f 0 13
4.03 f 0 30
5 08 f
Preperfusion period, nonworking (n = 5) Control, working heart nonligated (n = 10)’ Control, ligated (n = 9) Metoprolol (n = 6) Diltiazem (n = 6) l
ET AL
Tissue High Energy Phosphate Contents in the lnfarcting and Noninfarcting Myocardium -___ _ Adenosine Triphosphate (hmol/g Fresh Weight)
Data + p
INFARCTION-HAMM
2.83 Jo 0.30 3.38 & 0 29 3.70 f 0 13+
0.60 f 0.05 1.08 f 0.17+ 0.97 f 0 12+
4.01 f 0 46 3.95 f 0.28 3.70 f 0.27
lnfarcting
1.21 f 0.11 1.72 f 0.23+ 1.46 f 0.14
from Kannengiesser et aLis 05. 01 versus control, ligated are mean f standard error of the mean.
zones of the heart in this model.18 However, even considerable admixture of ischemic and nonischemic tissue in the assay samples could not explain the more beneficial effect of diltiazem than metoprolol on adenosine triphosphate levels. Only metoprolol improved phosphocreatine values in the ischemic zone. Discussion Cellular damage: The present investigation demonstrated that the beta-adrenoceptor antagonist metoprolol and the slow channel inhibitor diltiazem exert substantial protection against cellular damage on acutely infarcting myocardium. Similar results have been obtained for other compounds in each drug group,8m1fi although not consistently.27-29 In the concentrations used, both interventions were equally effective in protecting myocardial cell integrity as assessed by enzyme loss in our preparation, and in addition preserved adenosine triphosphate in the infarcting tissue. Metoprolol, in addition, maintained phosphocreatine levels (Table II). However, the 2 agents exhibited opposing effects on the mechanical function of the heart after coronary ligation. Compared with diltiazem metoprolol caused deterioration of cardiac output and left ventricular work and efficiency immediately after production of acute myocardial ischemia. Effect on mechanical function: Some studies in larger animal9 have not revealed a depressant effect of beta-adrenoceptor antagonism on the mechanical function of the heart, but the compound used was practolol, which showed little cardiodepressant effect in some patient studies. A possible explanation for the relative absence of cardiodepressant effects by practolol is the intrinsic sympathomimetic activity that metoprolol does not possess. The concentration of metoprolol we used was probably higher than comparable doses of other beta-adrenoceptor antagonists used in most intact animal studies. In patients with acute infarction, intravenous metoprolol has also been used without precipitating left ventricular failure,31 but the patients initially had no clinical features of congestive heart failure, and therefore probably had relatively small infarcts. Ligation of the left main coronary artery in rats results in an infarct involving almost all of the
free wall of the left ventricles* and with a mean overall reduction of coronary flow to 57% of the preligation value.18 Therefore, the more extensive size of the ischemic zone in this preparation probably produced a more significant cardiac deterioration than that found in most patients with acute infarction. Improvement of the mechanical function of the heart mediated by diltiazem or other slow channel inhibitors has also been demonstrated in ischemic-reperfused and has been further confirmed in the preparations,33-s5 case of nifedipine by measurements of enhanced regional myocardial contractility in the ischemic myocardium of larger animals. 36 The effect on mechanical performance is considered to be related to the ability of these compounds to prevent deleterious intracellular calcium accumulation which can impair left ventricular contractile force and relaxation,37 but improved coronary perfusion to the ischemic zone is likely to be another important factor.ss,38 Verapamil, in contrast, appears to depress regional myocardial contractility in the ischemic but not the nonischemic myocardium, whereas propranoloi depresses both normal and ischemic myocardium.3g Preservation of cell integrity: A variety of mechanisms have already been proposed for the reduction of cellular damage in the acutely infarcting myocardium by beta-receptor antagonists and slow channel inhibitors.3,s,8J2 Our experiments show that protection can be achieved when heart rate is not reduced. Severity of cellular damage has been assessed by release of lactate dehydrogenase and the tissue content of high energy phosphates. It has to be noted that phosphocreatine, increased in the ischemic zone only by metoprolol, cannot be related to the extent of enzyme release40 in contrast to the level of adenosine triphosphate.41 It may be speculated that the elevated adenosine triphosphate content achieved by both metoprolol and diltiazem in ischemic myocardium could represent a specialized pool to which Bricknell and Opie*2 attributed a role in protecting the cell membrane. Myocardial perfusion: In addition to the beneficial effects of diltiazem on the calcium homeostasis in the ischemic myocardium, its vasodilating activity has to be considered as a mechanism of protection.saJs Dilti-
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LV work+
4 LV
INFARCTION-HAMM
work
j . .. tissue I’ ATP 4 +coronary
flow
+ LDH-rel:ase f
FIGURE 4. Comparative effects of metoprolol and diltiazem on acutely infarcting myocardium. Results are based on comparisons with control hearts in Tables I and Il. ATP = adenosine triphosphate; LDH = lactic dehydrogenase; LV = left ventricle
azem causes an increase in the blood flow in the peripheral zone of regional myocardial ischemia in the pig mode1.43T44 Because no evidence for a coronarv “steal” phenomenon43-45 with diltiazem has been reported (in contrast to nifedipine27), one may suppose from our experiments that there was a redistribution, which was not clearly detectable by measuring the total coronary flow rate. By this, oxygen supply to the ischemic tissue would be imnroved and tissue acidosis diminished through washout of lactate and hydrogen ions. Excess hydrogen ions not only interfere with key reactions of metabolic pathways, 46 but also represent another factor that has been associated with impairment of the contractile function.47. Even though metonrolol exhibited reduced total coronary flow-rates in our model, we cannot completely exclude a favorable redistribution of flow to the ischemic zone as found in the pig mode1.48 A reduced washout of enzymes is possible in the case of metoprolol, so that enzyme release was falsely low; this sequence is rendered unlikely by increased values for high energy phosphates in the infarcting tissue. In addition, no facilitated washout is apparent for diltiazem (Fig. 2). Therefore, equal protection of cell integrity in the presence of significantly different flow rates suggests that mechanisms other than nerfusion effects are involved in mediating preservation of ischemic myocardium. However, the opposing effects on mechanical function may possibly be related to the degree of myocardial perfusion. Critique of the model: The isolated working rat heart with coronary artery ligation is a model with both advantages and disadvantages. The dynamic function of the heart can be studied under reproducible and controllable conditions.18 whereas the severitv of cellular damage induced by coronary artery ligation can be measured continuously and accurately by enzyme release.20 A limitation of this preparation is that it fails to allow physiologic interaction of the heart with the peripheral circulation. The preparation is severed from the autonomic nervous system, quite unlike the situation in patients with infarction. The results obtained with this model cannot directly be extrapolated to the human heart, because heart rate, cardiac output, and coronary flow rates of the isolated rat heart are not comparable to those in man. The situation in any animal .z
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model when healthy coronary arteries are abruptly ligated may not be relevant to the much more complex coronary artery anatomy in patients when occlusive disease can antedate acute infarction by many years. Another outstanding question is whether the beneficial effects of diltiazem administration before coronary artery ligation could also be achieved after the onset of the infarction process. However, most of the interventions initially applied before coronary artery occlusion1 have also been effective within the first hours after coronary occlusion.:~ The experimental concentrations of the 2 potentially cardioprotective agents are also open to criticism. With diltiazem, therapeutic concentrations in patients may reach 3 X lop7 g/ml or 7.3 X low7 M.5 Similar concentrations inhibit smooth muscle excitation coupling in the coronary arteries of the pig2 and increase spontaneous cycle length in the isolated sinoatrial node in the rabbit.” Hence, the concentration (4 x lop7 M) used in this study may well be of therapeutic importance in clinical practice. With metoprolol, a relatively large dose (10e4 M) was used that is well above the effective blood level of about 10e7 M.4g At such high concentrations, cardioselectivity might well have been lost, hence tending to reduce coronary flow by an unopposed alpha-mediated vasoconstriction. However, because our study was based on a comparison of 2 similarly potent protective concentrations, 10B4 M of metoprolol was required to be as effective as 4 X 10e7 M of diltiazem.23,24 This concentration of metoprolol was also required to antagonize isoproterenol lop6 M. The local concentration of norepinephrine in ischemic myocardium is likely to be about 10h6 M.40 At such very high concentrations, effects other than beta-antagonism, such as membrane protection, are likely to be relevant. Such comparisons apply strictly to the isolated heart. In patients with acute myocardial infarction, circulating catecholamine concentrations (largely norepinephrine) are increased to about 5 X low9 Mso; such concentrations of norepinephrine fail to increase enzyme release from the isolated rat heart.40 Hence, our data may possibly have application in acute myocardial infarction even in the absence of added catecholamine stimulation. Conclusion: The present study demonstrates that beta-adrenoceptor antagonism by metoprolol at large doses and slow channel inhibition by diltiazem at apparently therapeutic doses are equally effective in reducing cellular damage in the acutely infarcting myocardium but that there are opposing effects on the mechanical function of the heart. Diltiazem offered substantial cardioprotection in the presence of significantly better mechanical performance than metoprolol. Whereas metoprolol reduced cardiac output and left ventricular work compared with untreated control hearts, these variables appear to be influenced in a favorable way by diltiazem. Although our results were obtained in an isolated, perfused rat heart model, the principles involved could be of considerable importance in a clinical setting. Patients with acute myocardial infarction often have restricted ventricular function, Volume 50
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especially when the infarct is large, as in our model.“’ Further myocardial depression by beta-receptor antagonism could initiate cardiac decompensation and result in cardiogenic shock, whereas our studies raise the expectation that diltiazem could improve the mechanical function possibly by facilitating blood flow to the ischemic zone. Although conclusions drawn from diltiazem may not apply to other slow channel inhibitors and evidence indicates that further experimental data in other models are required, both theoretical and experimental data suggest that slow channel blockade could become a more promising therapeutic approach than beta-adrenoceptor antagonism in minimizing myocardial infarct size
(Fig. 4). Acknowledgment Dr. C. Hamm thanks the Chris Barnard Fund and the Deutsche Forschungsgemeinschaft for generous support. This work forms part of an ongoing program of the Medical Research Council of South Africa, who are also thanked for support. We thank Professor W. Bleifeld for cooperation and advice. References 1. Maroko PR. Kjekshus JK, Sobel BE, et al. Factors influencing infarct size following experimental coronary artery occlusions. Circulation 1971;43: 67-82. 2. Fleckensteln A. Specific oharmacoloov of calcium in mvocardium. cardiac pacemakers, and.vascul& smooth n%scle. Ann Rev bharmacof Toxicol 1977:17:149-166. 3. Kloner RA, Braunwaid E. Observations on experimental myocardial ischemia. Cardiovasc Res 1980;14:371-395. 4. Opie LH, Thomas M. Propranolol and experimental myocardial infarctjon substrate effects. Postgrad Med J 1976;52(Suppl 4): 124-132. 5. Kawai C, Konishi T, Matsuyama E. Dkazaki H. Comparative effects of three calcium antagonists, diltiazem, verapamil and nifedipine on the sinoatrial and atrioventricular nodes. Circulation 1981:63:1035-1042. 6. Feriinz J, Easthope JL, Aronow WS. Effects of verapamil on myocardial performance in coronary disease Circulation 1978;12:28-33. 7. Marshall RJ, Parr&i JR. Comparative effects of propranolol and practolol in the early stages of experimental canine myocardial infarction. t3r J Pharmacol 1976;57:295-303. 9. Henry PD. Hucaieib R, Borda LJ, Roberts R, WIlliamson JR, Sobei BE. Effects of nifedipine on myocardial and ischemic injury in dogs Circ Res 1978;43:372-380. 9. Nagao T, Matlib MA, Frankiln D, Millard RW, Schwartz A. Effects of diltiazem, a calcium antagonist, on regional myocardial function and mitochondria after brief coronary occlusion. J Mol Cell Cardiol 1980;12:2943. IO. Relmer KA, Lowe JE, Jennings RE. Effect of the calcium antagonlst verapamil on necrosis following temporary coronary artery occlusion In dogs. Circulation 1977;55:581-587 11. Smith HJ, Singh BN, Nlsbet HD, Norris RM. Effects of verapamil on infarct size following experimental coronary occlusion. Cardiovasc Res 1975;9. 569-578. 12. Weishaar R, Ashikawa K, Bing RJ. Effect of diltiazem. a calcium antagonist, on myocardial ischemia. Am J Cardiol 1979:43:1137-l 143. 13. Wende W, Bieffeid W, Meyer J, Stuhiin HW. Reducbon of the size of acute experimental myocardial Infarction by verapamil. Basic Res Cardiol 1975;70:198-208. 14. Libby P, MarokoPR, Coveil JW, Mailoch Cl, Ross J Jr, Braunwaid E. Effect of practolol on the extent of myocardial ischemic in)uty after experimental coronary occlusion -. and its effects on ventricular function in the normal and lscnemlc neart. tiardiovasc Res 1973;7.167-173. 15. Miura M, Thomas R, Ganz W, et al. The effect of delay in propranolol administration on reduction of myocardial infarct size after experimental coronary artery occlusion in dogs Circulation 1979;59:1148-1157. 16. Reimer KA, Rasmussen MM, Jennings RB. Reduction by propranolol of my-dial necrosis following temporary coronary artery occlusion In dogs. Circ Res 1973;33:353-363 17. Neely JR, Liebermeiater H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart Am J Physiol 1967;212:804-814. 18. KannangiasserGJ, Lubbe WF, Opie LH. Experimental myocardial infarction with left ventricular failure in the isolated perfused rat heart Effects of lsoproterenol and pacing. J Mol Cell Cardiol 1975;7:135-151. 19. Van der Laarse A, Holiaar L, Van der Vatk LJM. Release of alpha hydfoxybutyrate from neonatal rat heart cell cultures exoosed to anoxia and reoxygenation. Comparison with impairment of strudture and function of damaged cardiac cells. Cardiovasc Res 1979;13.345-353.
VERSUS METOPROLOL IN EXPERIMENTAL
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20. De Leiris J, Opie LH. Effect of substrates and of coronary artery ligation on mechanlcal performance and on release of lactate dehydrogenase and creatlne phosphoklnase in isolated working rat hearts. Cardiovasc Res 1978.12:585-596 21. Kannengiesser GJ, Opie LH, van der Werff TJ. Impaired cardiac work and oxygen uptake after reperfusion of regionally lschemic myocardlum J Mol Cell Cardiol 1979,i 1’197-207 22. Frishman W, Silverman R. Cltmcal pharmacology of the new beta-adrenerglc blocking drugs Part 2 Physiologic and metabotic effects Am Heart J 1979,97:797-807 23. Saikawa T, Nagamoto Y, Arita M. Electrophysiologtc effects of diltiazem. a slow channel inhibltor, on camne cardiac fibers Jpn Heart J 1977;18: 235-245 24. Hamm CW, Opie LH. ProtectIon of lnfarctlng card&urn by slow channel inhfbrtors (abstr) Am J Cardiol 1982,49-942 25. Hamm CW, Opie LH. Erhaltung akut lschamlschen Myokards durch Diltiazem Amsterdam Excerpta Medica, In press 26. Opie LH, Mansford KRL, Owen P. Effects of Increased heart work on glycolysis and adenlne nucleotldes in the perfused heart of normal and diabetic rats Biochem J 197 1,124.475-490 27. Peter T, Heng MK, Singh BN, et al. Failure of htgh doses of propranolol to reduce experimental myocardlal lschemic damage Circulation 1978:57: 534-540 28. Karlsberg RP, Henry PD. Ahmed SA, Sobei BE, Roberts R. Lack of protectlon of ischemic myocardtum by verapamil in conscious dogs Europ J Pharmacol 1977,42 339-346 29. Selwyn AP, Welman E, Fox K, Horlock P, Pratt T, Keiin M. The effects of nifedlplne on acute experimental myocardial lschemla and infarction in dogs. Circ Res 1979;44 16-23. 30. Libby P, Maroko PR, Covell JW, Malloch Cl, Ross J Jr, Braunwald E. Effect of practolol on the extent of myocardial ischemic injury after experimental coronary occlusion and its effects on ventricular function in the normal and lschemic heart Cardiovasc Res 1973,7.167-173 31. Waagstein F, Hjalmarson A. Double-blind study of the effect of cardloselective beta-blockade on chest paln in acute myocardlal infarction Acta Med Stand 1976,587(Suppl).201-208. 32. Fishbein MC, Hare CA, Gissen SA, Spadaro J, Maclean 0, Maroko P. Identification and quantification of histochemical border zones during evc$tlon of myocardial infarctlon in the rat Cardlovasc Res 1980;14:4133. Jolly SR, Menahan LA, Gross GJ. Diltiazem in myocardlal recovery from global tschemia and reperfuslon. J Mol Cell Cardlol 1981;13:359-372. 34. Weishaar RE, Bing RJ. The beneficial effect of a calcium channel blocker, ddtiazem. on the ischemlc reperfused heart J Mol Cell Cardiol 1980;12: 993-1009 35. Sherman LG, Liang CS, Boden WE, Hood WB. The effect of verapamil on mechanical performance of acutety ischemic and reperfused myocardium In the conscious dog Circ Res 1981;48:224-232. 36. Henry PD, Shuchleib R, Clark RE, Perez JE. Effect of nifedipine on myocardlal ischemla analysis of collateral flow, oulsatile beat and reaional muscle shortening Ati J Cardiol 1979;44.817’-824 37. Henry PD, Shuchleib R, Davis J, Weiss ES, Sobel BE. Myocardial contractufe and accumulation of mltochondrlal catclum in lschemic rabbit heart Am J Physrol 1977;233:H677-684. 38. Jolly SR, Gross GJ. Improvement of lschemlc myocardlal blood flow following a new calcium antagonist Am J Physiol 1980;239,Hl63-HI71 39. Smith HJ, Goldstein RA, Grlffith JM, Kent KM, Epstein SE. Regional contractiltty Selective depression of lschemtc myocardium by verapamil. Circulation 1976.54:629-635. 40. Waldenstrom Ak, Hjalmarson AC, Thorneli L. A possible rote of noradrenaline In the development of myocardial Infarction Am Heart J 1978.95:43-5 1, 41. Gebhard MM, Denkhaus H, Sakai K, Spleckermann PG. Energy metabolism and enzvme release J Mol Med 1977.2 271-783 -_-. 42. Brickneil OL, Opie LH: Effectsof &b&aies on tissue metabohc changes in the Isolated rat heart during underperfusion and on release of lactate dehydrogenase and arrhythmias during reperfusion Circ Res 1978;43: 102-115 43. Millard RW. Changes in cardiac mechanics and coronary blood flow of regtonally lschemic porcine myocardium Induced by diltiazem Chest 1980:78(Suppl 1):193-199 44. Franklin D, Millard RW. Nagao T. Responses of coronary collateral flow and dependent myocardial mechanical function to the calcium antagonist, diltiazem Chest 1980;78(Suppl 1),200-204 45. Nagao T, Murata S, Sat0 M. Effects of diltiazem (CRD-401) on developed coronary collaterals in the dog Jpn J Pharmacot 1975;25:281-288, 46. Opie LH. Metabolic regulation In ischemia and hypoxia. Effects of regional tschemla on metabolism of glucose and fatty acids Circ Res 1976;38(Suppl 1) l-52-l-74. 47. Williamson JR, Schaffer SW, Ford C, Safer B. The cellular basis of ischemia and Infarction. Contribution of tissue acidosis to ischemic injury in the perfused rat heart Circulation 1976;53jSuppl 1):1-3-i-14. 48. Muller CA, Hamm CW, Opie LH, Pelsach M, Glhwala D. Effect of betaadrenoceptor antagonism on regional left ventricular blood flow and incidence of ventflcular fibrillation after coronary artery ligation in pigs (abstr). Proc 8th Int Cong Pharmacol. Tokyo, 1981 49. Regardh CG, Johnsaon G, Jordo L, Lundborg P, Persson BA, Ronn 0. Plasma concentrations and beta-blocking effects in normal volunteers after tntravenous doses of metopfolol and propranotol J Cardiovasc Pharmacol 1980;2:715-723. 50. Vetier NJ, StrangeRC, Adams W, Oliver MF. lnitlat metabolic and hormonal response to acute myocardial infarction Lancet 1974;1:284-289. 51. Bleifeld W, Mathey D, Hanrath P, Buss H, Effert S. Infarct size estimated from serial serum creatine phosphokinase in relation to left ventricular hemodynamics Circulation 1977.55:303-311
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