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The beneficial effect of a calcium channel blocker, diltiazem, on the ischemic-reperfused heart
The Beneficial Diltiazem, RONALD
Effect of a Calcium Channel on the Ischemic-Reperfused E. WEISHAAR
AND RICHARD
Blocker, Heart
J. BING
Institute of Applied Medical Research and Huntington Memorial Hospital, 100 Congress Street; Pasadena, Galajknia 91105, and the California Institute of Tec~n~~~~, Division of Chemistry and Chemical Eng~~er~ng, Pasadena, ~a~~~ar~~~, U.S.A.
Huntington
(Received 15 Afovember
1979; accepted >n revisedform
25 March
1980)
R. E. WIXSHAAR AND R. J. BING. The Beneficial Effect of a Calcium Channel Blocker, Diltiazem, on the Ischemic-Reperfused Heart. Journal of Molecular and Cellular Cardi&%s (1980) 12,993-1009. Diltiazem-HCl,@ a potent calcium channel blocker, was found to be effective in moderating the harmful effects of reperfusion on the severely ischemic myocardium. In isolated working rat heart preparations it was found that 120 min of giobal &hernia followed by 15 min of reperfusion resulted in a massive leakage of creatine phosphokinase into the coronary effluent, and in many cases, in fibrillation and/or contracture. Left ventricular end-diastolic pressure increased sharply during reperfusion in these hearts. Reperfusion did not affect tissue ATP levels, but did increase creatine phosphate somewhat. When Diltiazem was added to the per&sate (final concentration 0.4 PM) 5 min prior to re-establishment of flow, the deleterious effects of reperfusion were greatly reduced. None of the hearts fibrillated on reperfusion, and none developed contracture. Left-ventricular end-diastolic pressure was increased only slightly in these hearts. The amount of creatine phosphokinase released into the coronary effluent during m-flow was only one-half that which was released by hearts reperfused in the absence of Diltiazem. In the Diltiazem-treated hearts reperfusion restored creatine phosphate to near-normal levels, although ATP levels were not increased. The beneficial effects of Diltiazem are probably related to its ability to reduce the rapid and massive mitochondrial calcium overloading which normally accompanies reperfusion of severely ischemic myocardium.
KEY WORDS: Calcium channel blockers; Global ischemia; Reperfusion; Enzyme release; High energy phosphates; Left ventricular pressure.
Contracture;
1. Introduction The efficacy of reperfusing ischemic tissue is currently a controversial subject [4, 8, 23,291. Studies have shown that in a number of instances abrupt revascularization of the myocardium leads to a paradoxical extension of the damage due to &hernia alone [5, 6, 2.5, 401. The biochemical basis for this finding is not presently known, but is apparently related to the inability of the severely ischemic myocardium to maintain normal calcium homeostasis during reperfusion [14, 411. Shen and Jennings were the first to demonstrate that a sudden restoration of Aow Address for reprints: Richard J. Bing, M.D., Street, Pasadena, CA 91105, U.S.A. 0022~2828/80/ 100993+ 17 $02.00/O
Huntington
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0 1980 Academic
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Press Inc. (London)
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leads to a rapid and massive influx of calcium ions into the ischemic region, which are subsequently accumulated within the mitochondrial matrix and precipitated as calcium phosphate [36]. This influx coincides with a large release of creatine phosphokinase from the myocardium and is, according to Jennings, responsible for the development of contracture bands in the ischemic-reperfused region
[201. It therefore seemed appropriate to examine the effectiveness of a new calcium channel blocker,* Diltiazem-HCl, in reducing reperfusion-induced damage to the ischemic myocardium. The precise mechanism by which Diltiazem alters calcium influx is unclear. Saikawa et al. have indicated that Diltiazem acts as an inhibitor of the transmembrane “slow channel” through which calcium ions enter the cell [33]. However, other recent studies on the effect of Diltiazem on smooth muscle have suggested that Diltiazem might act instead through inhibition of the regenerative release of activator calcium from the internal stores [X, 271. Diltiazem has also been shown previously by Weishaar et al. to diminish the rate of ATP hydrolysis and to decrease fatty acid and lactate levels in the ischemic myocardium [39]. Nagao and co-workers also found that Diltiazem administration prevented the depression of state 3 respiration in mitochondria normally observed following brief periods of ischemia [27]. In the present study the effects of reperfusion following a lengthy period of ischemia were examined using isolated, working rat hearts, in which the degree of flow reduction and other experimental conditions could be closely controlled. The results demonstrate that Diltiazem, when present in the circulating perfusate, significantly reduced the deleterious effects of reperfusion on the ischemic myocardium. 2. Materials
and Methods
Exferimental
model
Male Sprague-Dawley rats, weighing 250 to 400 g each and fasted overnight prior to the experiment, were used throughout the study. The animals were i.p.) and then injected intraanesthetized with Nembutal (40 mg/ animal, venously with heparin (2.5 mg). Ten minutes after the administration of heparin the heart was rapidly excised and placed in a beaker of ice-cold saline until contractions ceased (20 to 30 s). The hearts were perfused according to the working heart model described by Neely and Rovetto [29]. The entire system was water-jacketed and the temperature maintained at 37°C. After cannulating the aorta a preliminary washout is a more suitable term than “calcium “calcium channel blocker” * According to Katz, antagonist” since such agents are “known to ‘antagonize’ Ca2+ only at specific sites in the sarcolemma (the slow channel) and are virtually without effect on a variety of Ca2+-binding and transport systems” 1211.
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perfusion was begun from a reservoir located 60 cm above the heart. The pcrfusate consisted of Krebs-Hens&it buffer containing 11 mu glucose and 10 mg;‘l heparin, and was bubbled xvith 1)5?; 0, : S?; (D,. (Heparin bvas nor present in the medium during the subsequent working perfusion.) ‘l’hc heart was perfused in this manner for 10 min, during kvhich time the left atrium \cas cannulated via the pulmonary veins. Once the atrium was cannulated, flo\~ from the ~~ashout reservoir wxs stopped and the heart immediately placed in a working mode ivith the left atria1 Ming pressure set at 10 cm H,O and the afterload at 60 mmHg hydrostatic pressure. The perfusate { 130 ml) was thereafter continuously recirculated, although at 30 min intervals 50 ml were removed and an equal amount of f&h perfilsate MXS added to the oxygenation chamber of the apparatus. The ticarLs \vcrc electricall) paced at 225 to 275 beats/min, with the pacing voltage set at roughly lo”,, above the threshold level. ‘1%~ hearts were pcrfilsed in this fashion I‘or an additional 10 min before ischemia was begun. During this pre-ischcmic period hernodynamic measurements lvcrc made scvcral times to ensure the stability of the preparation before comrncncing the expcrimcnt. (:oronary flow was reduced by the placement of a one-\yay valve in the aortic outflow tract which effectively prevented retrograde perfusion of the coronary arteries during diastole (for a detailed drawing of the one-\vay valve see Neely and Iiovctto 1291). During the prc-ischemic and also the reperfusion phases of the cxpcrirncnt, coronary flow was diverted around this one-way valve by means of a bypass tube. Ischemia was induced by clamping the bypass tube and f‘orcing the aortic outflow to pass through the one-way valve. Following this maneuver vcntricular pressure dcvclopmcnt quickly began to decline, \vith peak systolic pressure being reduced by roughly 80’1~ of the pre-ischemic Ici~cl lvithin 10 min of the onset of ischcmia. Coronary flow declined by 80 to 900,,, during this period and thercafter fell to roughly 5T.b of the normal level. Low-flow ischcmia \vas maintained for a total of 120 min, at which time lhe hearts were reperfuscd for an additional 15 min (a number of hearts were not rcperfuscrl, and following 15, 30, 60, I20 or 135 min of ischcmia alone the experiment was terminated). A 2 h period of ischernia was chosen in the present study since preliminary experiments rcvealcd rhat reperfitsion following shorter periods of flow reduction ( 15 to 60 rnin) occasionally reversed the hcmodynamic and biochemical changes produced by ischernia, and rarely induced contracturc. Occasionally contractile activity ceased after 15 to 20 min of ischemia, or during the first minutes of reperf’usion. In most of these hearts this cessation could be rcvcrsed 1)~ increasing the pacing voltage slightly. I Io\\.ever, in a number of rtlc reperfused hearts cvcn extremely high pacing voltages could not elicit a contractilc response. In those hearts in which the effect of Diltiazem on reperfusion-induced damagewas studied, a 5 ml aliquot of fresh perfusate, in which an appropriate amount 01‘
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Diltiazem had been dissolved to provide a circulating concentration of 0.4 mM, was added to the oxygenating chamber of the perfusion apparatus 5 min prior to the onset of reflow. In all other experiments an equal amount of fresh perfusate alone was added at this time. Preliminary experiments in this laboratory have shown that at the concentration used in the present study, Diltiazem exerts a negative inotropic and chronotropic effect on the perfused nonischemic rat heart. In unpaced hearts, the heart rate was reduced by 40 to 60% within 3 min of the addition of Diltiazem to the perfusate, and in the paced heart left ventricle systolic pressure (LVSP) and left ventricular dP/dt,,, were reduced by 20 to 30% of normal levels. These effects were not permanent, however, and a partial restoration of myocardial contractility began approximately 10 to 15 min after Diltiazem administration. Hemodynamic measurements Aortic and atria1 pressures were monitored by conne&ng pressure transducers (Gould No. P23ID) to side-arms on the respective ccannulae. In the ischemic preparations, the aortic pressure was measured below the one-way valve. Intraventricular pressures were measured by inserting a 20-gauge needle, fluid-filled and connected to a pressure transducer, through the apex of the heart and into the left ventricle. All three transducers were,in turn connected to an Electronics for -flow was estimated by collecting the Medicine Monitoring system. Coronary effluent from the pulmonary artery in a graduated cylinder. Measurements of coronary flow were made every 5 min during the first 30 min of ischemia, and at 30 min intervals thereafter. Biochemical analysis In a number of experiments samples of the coronary effluent (0.2 ml) were collected in cooled glass vials at the following times: prior to the onset of ischemia, after 60 and 120 min ischemia, and after 1, 3, 5, 10 and 15 min reperfusion. These were then quickly frozen until analysis. (In experiments where the effects of ischemia alone were monitored, these latter five samples were collected following 121, 123, 125, 130 and 135 min ischemia). Creatine phosphokinase (CPK) (ATP-phosphotransferase) activity in these samples of effluent was determined by the method of Rokalski [32]. The rate of CPK leakage was then expressed in international units per minute per gram dry weight according to the method described by Ganote and co-workers [PI. The total amount of CPK released during the 15 min period of reperfusion (or during the corresponding period of ischemia) was determined by Simpson’s rule [Z]. In hearts in which high energy phosphate levels were determined the left ventricle .was, at the indicated times, rapidly frozen using stainless steel tongs precooled for several minutes in liquid nitrogen. The samples were then wrapped in
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aluminium foil and stored in liquid nitrogen until they could be analyzed. The levels of the following high energy phosphates were measured in a portion of the frozen ventricle : creatine phosphate (CP), adenosine-5’-triphosphate (ATP), and adenosine-5’-monophosphate (AMP). adenosine-5~-diphosphate (ADP) These levels were determined in perchloric acid extracts of the tissue samples according to standard procedures employing NADH-coupled reactions [3]. The remaining portion of the left ventricle was used to determine the dry weight of the heart. In the absence of ischemia, these preparations typically maintained stable function of periods of 2 h or longer, as determined both metabolically and hemodynamically. Coronary flow remained above 10 to 12 ml/min, and ATP and CP levels were only 9.6% and 8.3%, respectively, below zero-time levels after 2 h of perfusion (data not shown). Reagents The reagents used in these experiments were of an analytical grade quality. Solutions were made in all-glass distilled water. Diltiaz~m-HCl~ was kindly provided by Tanabe Seiaku Co., Ltd., Osaka, Japan.
Statistical analysis Data were evaluated using Student’s t test, or when necessary, an “a priori” t test, and the results were expressed as the mean & the standard error 1371. In the case where total CPK release during the period of reperfusion, or the corresponding period of ischemia, was evaluated, the results were expressed as the mean i the 95% confidence limits, and the significance was ascertained by both a standard two-way Analysis of Variance and a one-way Analysis of Variance after first transforming the data to its logarithmic equivalent [Z, 371.
3. Results The consequences of reperfusion on the severely ischemic myodardium, in both the presence and the absence of Diltiazem, will be divided into hemodynamic and the metabolic effects. Efects of reperfission oa myocardial Aemodynamics As can be seen in Figure 1, the decline in coronary flow during the 2 h period of global ischemia was rapid, falling to a level which was only 5% of the pre-ischemic value. Reperfusion only partially restored coronary Aow to a normal level. Coronary flow was, however, significantly higher (P < 0.05) in the Diltiazem
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FIGURE 1. Coronary flow in ischemic-only hearts (O---O), and in hearts reperfused in either the presence (m--m) or absence (A-A) of Diltiazem. Hearts were perfused in a working fashion with Krebs-Henseleit buffer containing 11 rn~ glucose at 37°C. Changes in flow are expressed as a percentage of the pre-ischemic level. Each symbol represents the mean & S.E. of at least seven experiments. * Significantly (P < 0.05) .mcreased as compared to percentage change in corononary flow following a corresponding period of &hernia; 7 significantly (P < 0.05) increased as compared to percentage change in coronary flow in hearts reperfused in the absence of Diltiazem.
treated hearts than in the hearts reperfused in the absence of the drug, indicating that Diltiazem had a vasodilating effect on the coronary arteries (Figure 1). Global ischemia also produced a prompt decline in the left ventricular systolic pressure (LVSP), and a corresponding increase in left ventricular end-diastolic pressure (LVEDP) [Figure 2(a) and (b)]. Th ese changes reached their maximum after approximately 15 min of ischemia and remained relatively constant thereafter. Aortic and atria1 pressures also diminished during the period of ischemia. As can be seen in Figure 2(a), reperfusion in the absence of Diltiazem led to a resulting in many cases from the marked increase in end-diastolic pressure, development of contracture. In these hearts LVEDP more than doubled within the first few minutes of reperfusion and remained at this elevated level throughout the period of reflow. Systolic pressure increased during reperfusion as well [Figure 2(b)]. This increase was also a reflection of contracture development and did not represent a reversal of the effects of ischemia on myocardial contractile function. In the Diltiazem-treated hearts these deleterious effects of reperfusion onhemodynamic function were virtually eliminated [Figure 2(a) and (b)]. LVEDP did not rise during the period of reperfusion and, in some cases, was restored to
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near the pre-ischemic level. LVSP rose slightly during reperfusion, though the increase differed from that which was seen in the non-Diltiazem hearts in that the increase was not related to the development of contracture. The effects of ischemia and reperfusion on the incidence of contracture development are described in Table 1 (a) and depicted schematically in Figure 3. In the present study contracture was defined as the absence of contractile activity (systolic pressure = end-diastolic pressure) accompanied by a rise in the enddiastolic pressure of at least 150% of the pre-ischemic level. This definition is equivalent to that used to define contracture clinically [23, 421. Contractile activity frequently ceased on reflow, occasionally even in the Diltiazem-treated hearts, though the cessation could often be reversed by increasing the pacing voltage. Hearts in which contractile activity could be restored in this way were not designated as having developed contracture. As can be seen in Table 1 (a), none of the hearts in which Diltiazem was added to the circulating perfusate developed contracture during reperfusion, while 37.5% (six of 16) of the hearts reperfused in the absence of Diltiazem eventually went into a state of contracture. These latter hearts also experienced a much greater incidence of fibrillation during reflow. In Figure 3 tracings obtained from four representative experiments are shown. In two of the experiments Diltiazem was added to the perfusate and in two it was not. It can be seen that reflow in the absence of Diltiazem resulted in rapid contracture in one experiment, and in the other in a slow deterioration of contractile function leading ultimately to the development of contracture. In the Diltiazem-treated hearts reperfusion was found to have little if any effect on contractile function in one experiment, and in the other resulted in a partial reversal of the effects of ischemia on contractility. In the latter experiment LVEDP was eventually restored to near the pre-ischemic level and LVSP to approximately 50% of normal (Figure 3).
Effects
of
reperfusion on myocardial metabolism
Enzyme release Figure 4 illustrates the effects of ischemia and of ischemia followed by reperfusion on creatine phosphokinase (CPK) leakage into the coronary effiuent. Ganote has previously shown that enzyme release by the myocardium is most likely related to and a number of studies have shown increased cell membrane permeability [II], marked ultrastructural damage accompanying the release of CPK [9, 13, 221. In the present study only small amounts of CPK were released by the myocardium during the period of global ischemia (Figure 4). However, reperfusion, in the absence of Diltiazem, was found to trigger a sudden, massive release of CPK, (g dry wt)-i following 10 min of which reached a peak of 14.3 & 2.7 iu mini reflow. Even after 15 min of reperfusion considerable quantities of CPK were still being released by the non-Diltiazem hearts (12.5 f 2.6 iu min-l (g dry wt)-r)
+ 103.4 f
f
-86.0
limits
95% confidence
37.5%
144.1*?
f
4 6.0%
(77.9 to 266.6)
+205.1
-63.5
37.5%
6
16
120 min ischemia 15 min reperfusion
release
*
5.5%
(29.7 to 165.5)
70.1”
+ 118.4 & 22.2%
-71.5
0
0
12
120 min ischemia 15 min reperfusion ( + Diltiazem)
(P < 0.05) increased as compared to the total amount of CPK released during the corresponding period of &hernia. (P < 0.05) increased as compared to the total amount of CPK released from hearts reperfused in the presence of Diltiazem.
2.2 (1.1 to 4.2)
(g dry wt)-r (15 min (or final 15 min
7.6%
0.5%
alone
and CPK
(b) CPK release iu CPK released reperfusion)-r ischemia)
of of
End-diastolic pressure (percentage pre-ischemic level at conclusion experiment)
of
0
0
13
Ischemia
development
Systolic pressure (percentage of pre-ischemic level at conclusion experiment)
contracture
contracture
on contracture
of hearts developing
Number
Percentage
of hearts examined
of hearts developing
Number
(a) Contractwe development
1. The effect of reperfusion
* Significantly t Significantly
TABLE
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R. E. WEISHAAR Im reflow
120m ischemia
2m reflow
AND 3m reflow
R. J. SING 4m reflow
5m reflow
IO m reflow
15m reflow
63 ss Z? Oi
-
FIGURE 3. Pressure tracings from four representative experiments. The hearts were perfused as described in Figure 1, In the lower two experiments a 5 ml aliquot of fresh pet-I&ate, into which an appropriate amount of Diltiazem had been dissolved to provide a final concentration of 0.4 nlM, was added to the circulating perfusate 5 min prior to the onset of reperfusion. In the upper two experiments an equal amount of fresh pet-f&ate alone was added. The tracings indicate the level of left ventricular pressure following 120 min of ischcmia, and 1,2,3,4,5, 10 and 15 min of reperfusion.
(Figure 4). The total amount of CPK released during the 15 min period of reperfusion averaged 144.4 IV/g dry wt, as compared to an average of2.2 W/g dry wt during the corresponding period of global ischemia [Table 1 (b)]. Diltiazem did not entirely prevent the reperfusion-induced release of CPK by the myocardium, although its presence in the perfusate during reflow did contribute to a significant reduction (P < 0.05) in the total amount of enzyme released, and therefore presumably to a decrease in membrane and ultrastructural damage. As can be seen in Figure 4 and Table 1 (b), the total amount of enzyme released from the myocardium was only about half of that which was released by the hearts which were not treated with Diltiazem (70.1 iu (15 min)--r (g dry wt)p’ vs. 144.1 iu (15 min)-1 (gm dry wt)-r) [Table l(b)].
Energy metabolism Global ischemia led to a prompt reduction in tissue levels of ATP and creatine phosphate (CP), while at the same time increasing, transiently, the level of AMP
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FIGURE 4. Myocardial enzyme release from ischemic only hearts ((II-O), and from ischemic hearts reperfused in either the presence (a-m) or absence (A-----A) of Diltiazem. Hearts were perfused as described in Figure 1, and samples of coronary effluent were taken at the following times: after the 10 min non-ischemic control period, after 60 and 120 min of &hernia, and after 1, 3, 5, 10 and 15 min of reperfusion (or after the corresponding period of &hernia). Each symbol represents the mean + LE. of five to seven experiments. The total amount of enzyme released during reperfusion was found to be significantly lower when .Diltiazem was included in the perfusate during reperfusion than when it was not, according to both methods of statistical analysis described in the text.
5(a) and (b)]. ADP levels remained fairly constant throughout the 120 min period of ischemia [Figure 5(b)]. In the absence of Diltiazem, reperfusion had little effect on myocardial energy stores. ADP did decrease significantly during reperfusion, but CP, ATP and AMP were not significantly altered [P’igure 5(a) and (b)]. Reperfusion in the presence of Diltiazem likewise had little if any effect on the level of ATP. However, reflow in the Diltiazem-treated hearts did produce a large and highly significant increase in the level of creatine phosphate from (5.5 j_: 1.7 pmol/g dry wt to 17.6 f 2.3 pmol/g dry wt) and a corresponding large decrease in AMP (from 5.7 pmol/g dry wt to 2.9 f 0.2 @mof/g dry wt). ADP was also significantly reduced in these hearts, but not to the degree which AMP was. It is of interest to note that the tissue level of ATP apparently had very little bearing on the development of contracture in the present study. As can be seen in Figure 5(a), ATP levels were essentially the same in hearts reperfused in either the presence or absence of Diltiazem (and for that matter in those hearts rendered ischemic for 30, 60 or 120 min), but only in those hearts in which Diltiazem was omitted from the perfusate during reperfusion did contracture occur (Table 1 (a)). [Figure
M.C.C.
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5 -I/-
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30
60
120
135 Time (min)
FIGURE 5. The effect of ischemia plus reperfusion on (a) ATP (a---@) and creatine phosphate (A---A) levels, and on (b) ADP (a---m) and AMP (+---+) levels. Hearts were perfused as described in Figure 1, and were freeze-clamped in sitz~ at the following times: after the lo-min non-ischemic control period, after 15, 30, 60 or 120 min of ischemia, or after 120 min of ischemia and 15 min of reperlnsion. In some experiments Diltiazem was added to the circulating perfusate 5 min prior to the onset of reperfnsion (open symbols). Each symbol represents the mean f S.E. of six to 10 hearts. * Significantly (P < 0.05) increased [Figure 4(a)] or decreased [Figure 4(b)] as compared to level following 120 min of &hernia. t Significantly (P < 0.05) to level in hearts reperfused in the increased [Figure 4(a)] or decreased [Figure 4(b)] as compared absence of Diltiazem.
4. Discussion Jennings and his colleagues were the first to report, in 1960, that reperfusion of ischemic myocardial tissue is not necessarily beneficial to the heart, and may in fact have the paradoxical effect of exacerbating the existing damage [I??]. The biochemical basis for this phenomenon is not known at the present time, but is thought to be related to changes which occur in the normal regulatory processes of the heart during ischemia, such that during reperfusion the injured cells rapidly take up calcium and other ions, leading to cell swelling and/or rupture. This uptake of calcium ions either causes it or is accompanied by marked changes in the myocardial ultrastructure, including the formation of contracture bands and separation of the basement membrane, as well as leakage of myocardial enzymes into the interstitial fluid [IO, 16, 22, 431. At the present time information is scarce concerning therapy designed to prevent the often harmful effects of reperfusion on the severely ischemic myodardium, although Ashraf and Bloor have shown that reperfusion with calcium-free plasma prevented the damage to the myocardial ultrastructure that otherwise accompanied reflow [I], and a recent preliminary report indicated that during ischemic arrest, Diltiazem might provide better protection for the myocardium than potassium [38]. In addition, Reimer et al. found that the necrosis which normally
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accompanies reperfusion in dogs was reduced by pretreatment with another calcium channel blocker, verapamil [30]. The results of the present study demonstrate that inclusion of Diltiazem-HCI in the perfusate during reperfusion significantly reduced the deleterious effect of reflow in the heart. The protective effect was demonstrated in several ways, including (i) prevention of contracture, which frequently (37.5:4 of the time) occurred when ischemic hearts were reperfused in the absence of Diltiazem; (ii), a significant reduction in the amount of CPK release into the coronary effluent during reflow (even when corrected for the vasodilating effect of Diltiazem) ; and (iii) restoration of myocardial creatine phosphate to near-normal levels. Since tissue levels of calcium were not measured in hearts reperfused In either the presence or absence of Diltiazem, it is impossible to say for certain that the protective effect of Diltiazem was due to a diminished influx of calcium ions into the ischemic-reperfused region. However, in the Diltiazem-treated hearts reperfusion produced a much smaller release of CPK than in the non-Diltiazem treated hearts and therefore presumably a reduction in the amount of ultrastructural damage. Previous studies have shown that such damage is related to a massive influx of calcium ions into the cell [19, 201. In addition, preliminary experiments in this laboratory have shown that Diltiazem, at the level used in the present study, diminished myocardial contractile force in the non-ischemic perfused rat heart (LVSP and left ventricular dP/dt,,, being reduced by 10 to 25% of normal levels). One of the striking findings was that reperfusion in the Diltiazem-treated hearts produced a marked increase in the tissue level of creatine phosphate (CP being restored to nearly two-thirds of the pre-ischemic value), while at the same time having little effect on the level of ATP [Figure 5(a)]. Similar quantitative discrepancies between creatine phosphate and ATP recoveries following reperfusion of ischemic tissue have been reported in several other studies [7, 17, 3.5, 40). Both IsseIhard and Chong found that the rapid restoration of tissue creatine phosphate levels normally precedes a similar, though slower, restoration of ATP levels as well [7, 171. In 1970, Gudbjarnason proposed the existence of separate mitochondrial and myofibrillar compartments of ATP, with creatine phosphate acting as a “transport form” of high energy bonds between the two [12]. Since that time Saks and his co-workers have provided evidence supporting this concept [34]. In the light of these findings, the results of the present study may be interpreted as indicating that, in the hearts reperfused in the presence of Diltiazem, there is a rapid restoration of mitochondrial synthesis of creatine phosphate, although there apparently exists a block in either the transfer of CP to the myofibrils, or in the reconversion of CP to ATP in that compartment. It is not possible to say from the information presented whether the block is permanent or only temporary. As was mentioned previously, it is interesting to note that the tissue level ofATP 2s2
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had little bearing on the development of contracture in the present experiments. ATP levels were essentially the same (5 to 6 pmol/g dry wt) in hearts reperfused in either the presence or the absence of Diltiazem; however, only in those hearts in which Diltiazem was omitted from the perfusate during reflow did contracture occur [Table 1 (a)]. Therefore, it would appear that the development of contracture was more likely related to an excessive influx of calcium ions into the nonDiltiazem treated hearts, than to a diminution in the cellular level of ATP. Other studies have also attributed the onset of myocardial contracture to disturbances in calcium homeostasis [23, 421. These findings differ from a recent report by Hearse et at., who found that contracture in isolated ischemic rat hearts was initiated once the tissue level of ATP fell below 12 pmol/g dry wt [IS]. There are several differences between the two studies, including the manner in which the hearts were rendered ischemic and the fact that in Hearse’s study the hearts were not paced, and would therefore arrest soon after the onset of ischemia, thus maintaining higher levels of ATP than would be otherwise expected. In the present study hearts occasionally arrested after 15 to 20 min of ischemia (at which time the tissue levels of ATP would probably be near the level described by Hearse ed rsl. as being critical). However, increasing the pacing voltage by 10 to 20% immediately restored and maintained contractile function for the remainder of the i,schemic phase, during which time the level of ATP continued to decline. None of the hearts developed contracture during the ischemic phase of the experiment. The results of this study demonstrate the highly protective effect of Diltiazem This protective effect is presumably on the ischemic-reperfused myocardium. related to the ability of this compound to restrict the previously described massive influx of calcium ions into the ischemic myocardium during reperfusion. In the present experiments it could be seen that revascularization, when combined with Diltiazem pre-treatment, resulted in a partial reversal of the effects ofischemia on the myocardium. Though the present study was restricted to an examination of only Dihiazem, other calcium channel blockers, such as nifedipine or verapamil, might also have proven beneficial in reducing the effects of reperfusion on the severely ischemic myocardium. However, since the mechanisms by which these three compounds inhibit the movement of calcium vary somewhat, the validity of such a general statement must await further evaluation.
The authors are grateful assistance, and to Mrs manuscript. The research reported
to Dr Patrick Pinto and Dr Gunter Pawlik Enid Soares for her help in the preparation in
this
paper
was
supported
by
grants
for their of the from
The
CALCIUM OHANNEL BLOCKERSAND Margaret the
W.
Council
and for
Herbert
Tobacco
Hoover,
Jr,
Foundation,
Research-U.S.A.,
Inc.,
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REPERFUSION Los New
Angeles,
York,
New
California
and
York.
REFERENCES 1.
ASHRAF, M., WHITE, I?. & BLOOR, C. M. Ultrastructural
influences of reperfusing dog with calcium-free blood after coronary artery occlusion. American Journal 90, 423--228 (1978). BARTSCH, H.-J. Taxhenbuch Mathematischer Formeln. Frankfurt/Main: Verlag Harri Deutsch (1976). BERGMEYER, H. V. Methods in Enqmatic Analysis. New York : Academic Press (1963). BRAUNWALD, E. Coronary bypass surgery at the crossroads. Xew England mourned of Medicine 297, 661-663 (1977). BRESNAHAN, G. F., ROBERT, R., SHELL, W. E., Ross, J. &. SOBEL, B. E. Deleterious effects due to hemorrhage after myocardial reperfusion. American 3ourncd of Cardiology 33, 82-86 (1974). BULKLEY, B. H. & HUTCHINS, G. M. Myocardial consequences of coronary artery bypass graft surgery: the paradox of necrosis in areas of revascularization. Circulation 56, 906-913 (1977). C~ONG, M. A., BEREZNY, G. M. & WINTON, T. L. Metabolism of the isolated perfused rabbit heart. I. Responses to anoxia and reoxygenation. II. Energy stores. Canad~un Jburnal of Physiological Pharmacology 56, 844-856 (1978). EFFLER, D. B. Myocardial revascularization: current state of the art. American Journal of Cardiolog, 36, 846-85 1 (1975). GANOTZ, G. E., SEBRA-GOMES: R., NAYLER, W. G. & JENNINGS, R. B. Irreversible myocardial injury in anoxic perfused rat hearts. American Journal of PathoEopy 80, 419-438 (1975). GANOTE, C. E., WORSTELL,~., IANNOTI, J. P. & KALTENBACH, J. P. Cell swelling and irreversible myocardial injury. American Journal ofpatholopy 88, 95-l 18 (1977). GANOTE, C. E. & KALTENBACH, J. P. Oxygen-induced enzyme release: early events and a proposed mechanism. JournaE of Molecular and Cellular Cardiology 11, 389-406 (1979). GVDBJARNSON, S., MATHES, P. & RAVENS, K. G. Functional compartmentation of ATP and creatine phosphate in heart muscle. 30~$~~~of Molec~lur and Cellular Cardiology 1, 325-339 (1970). HEARSE, D. J., HUMPHREY, S. M. & CHAIN, E. B. Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: a study of myocardial enzyme release. Journal of Molecular and Cellular Cardiology 5, 395407 ( 1973). HEARSE, D. J. Editorial: reperfusion of the ischemic myocardium. Journal o~~olec~~ar and Cell&r Cardiology 9, 605-616 (1977). HEARSE, D. J., GARUCK, P. B. & HUMPHREY, S. M. Ischemic contracture of the myocardium: mechanisms and prevention. Anerican Journal of Cardiology 39, 986-993 (1977). HEARSE, D. J., HUMPWREY, S. hiI. & BULLOCK, G. R. The oxygen paradox and the calcium paradox: two facets of the same problem? Journal of Molecular and Ce&lar Cardiology 10, 641-668 (1978). ISSELHARD, W. Experimental ischemia: total ischemia of the heart (metabolic recovery from complete anaerobiasis). In Brain and Heart Infar& pp. 125-136. K. J. myocardium o~Putho~o~
2. 3. 4. c5 .
6.
7.
8. 9.
10. 11.
12.
13.
14. 15.
16.
17.
1008
18.
19.
20. 21.
22.
23. 24. 25. 26. 27.
28.
29.
30. 31. 32. 33. 34.
R. E. WEI%HAAR
R. J. BING
Zulch, W. Kaufmann, K.-A. Hossmann & V. Hossmann, Eds. Berlin: SpringerVerlag (1977). JENNINGS, R. B., SOMMERS, H. M., SMYTH, G. A., FLACK, H. A. & LINN, H. Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog. Arc&es of Pathology 70, 68-78 (1960). JENNINGS, R. B., GANOTE, C. E. & REIMER, K. A, Ischemic tissue injury, American Journal of PatholoQ 81, 179-198 (1975). JENNINGS, R. B. Relationship of acute ischemia to functional defects and irreversibility. Circulation 53, Suppl. 1; 26-29 (1976). KATZ, A. M. & REUTER, H. Cellular calcium and cardiac cell death. American Journal of Cardiology 44, 1888190 (1979). KLONER, R. A., GANOTE, C. E., WHALEN, D. A. &JENNINGS, R. B. Effects of a transient period of ischemia on myocardial cells. II. Fine structure during the first minutes of reflow. American Journal of PatholoD 74, 3991122 (1974). KURKJI, H. S., BUCKBERG, G., MALONEY, J. V. & DELAND, E. C. Prevention of ischemic contracture (stone heart). Surgical Forum 24, 146-148 (1973). MCINTOSH, H. D. & GARCIA, J. A. The first decade of aortacoronary bypass grafting, 1967-1977. A Review. Circulation 57, 403-431 (1978). MONTOYA, H. D., MALET, J., PIFARIZE, R., BRYNJOLFSSON, G., MORAN, J. M., SULLIVAN, H. F. & GUNNAR, R. M. Hemorrhagic infarct following myocardial revascularization. Journal of Thoracic and Cardiouascular Surgery 75, 206-2 12 ( 1978). NABATA, H. Effects of calcium-antagonist coronary artery vasodilators on myocardial contractility and membrane potentials. Jafanese Journal of Pharmacology 27, 239-249 (1977). NAGAO, T., MATLIB, M. A., FRANKLIN, D., MILLARD, R. W. & SCHWARTZ, A. Effects of Diltiazem, a calcium antagonist, on regional myocardial function and mitochondria after brief coronary occlusion. Journal of Molecular andcellular Cardiology 12,29-44 (1980). NAKAHIMA, H., HOSHIYAMA, M., YAMASHITA, K. & KIYOMOTO, A. Effect of Diltiazem on electrical and mechanical activity of isolated cardiac ventricular muscle of guinea pig. Japanese Journal of Pharmacology 25, 383-392 (1975). NEELY, J. R. & ROVETTO, M. J. Technique for perfusing isolated rat hearts. Methods in Enzymology 39, 43-61 (1975). REIMER, K. A., LOWE, J. E. & JENNINGS, R. B. Effects of the calcium antagonist Verapamil on necrosis following temporary coronary artery occlusion in dogs. Circulation 55, 581-587 (1977). ROBERTS, R. & SOBEL, B. E. Coronary revascularization during evolving myocardial infarction-the need for caution. Circulation 50, 867-870 (1974). ROSKALI, S. B. An improved procedure for serum creatine phosphokinase determination. Journal of Laboratory and Clinical Medicine 69, 696-704 ( 1967). SAIKAWA, T., NAGAMOTO, Y. & ARITA, M. Electrophysiologic effects of Diltiazem, a new slow channel inhibitor, on canine cardiac fibers. Japanese Heart Journal 18,235-245 (1972). SAKS, V. A., ROSENSHTRAUKH, L. V., UDROVINAS, A. I., SMIRNOV, V. N. & CHAZOV, E. I. Studies of energy transport in heart cells: intracellular creatine content as a regulatory factor of frog heart energetics and force of contraction. Biochemical Medicine
16, 21-36 35.
AND
(1376).
SHARMA, G. P., VARLEY, K. G., KIM, S. W., BARWINSKY, N. S. Alterations in energy metabolism and ultrastructure ischemic myocardium after coronary occlusion. American 243 (1975).
J.,
COHEN, M. & DHALLA, upon reperfusion of the Journal of Cardiology 36, 234-
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36. 37. 38.
CHANNEL
BLOCKERSAND
REPERFUSION
1009
SHEN, A. C. &JENNINGS, R. B. Kinetics of calcium accumulation in acute myocardial ischemic injury. ,American Journal of Pathology 67, 441-452 (1972). SOKAL, R. R. & ROHLF, F. J. Biometry. San Francisco: W. F. Freeman and Co. (1969). VOUHE, P. R., HELIAS, J. & GROUMN, C. M. Myocardial protection through cold cardioplegia with potassium and Diltiazem (abstract). Circulation 60, Suppl. II;
240 (1979). 39.
40. 41.
42. 43.
WEISHAAR, R., ASHIKAWA, K. & BING, R. J. Effect of Diltiazem, a calcium antagonist, on myocardial ischemia. American ~a~r~al Q~Card~~~o~ 43, 1137-l 143 (1979). WEISHAAR, R., TSCHURTSCWENTHALER, G. V., ASHIKAWA, K. & BING, R. J. The relationship of regional coronary flow to mitochondrial function during reperfusion of the ischemic myocardium. Cardiology 64, 350-364 ( 1979). WRONG~MA~N, K. & PANA, S. D. Mit~hondrial calcium overload: a general mechanism for cell necrosis in muscle lesions. Lancet i, 672-674 (1976). WUKASCH, D. C., REUL, G. L., MILAM, J.D., HALLMAN,G. L. Sr Coorx~,D. A.The “Stone Heart Syndrome”. Surgery 72, 1071-1080 (1972). ZIMMERMAN, A. iY. E., DAEMS, W., H~~LSMANN, W. C., SNIJNER, J., WISSE, D. & DURRER, D. Morphological changes of heart muscle caused by successive perfusion with calcium-free and calcium-containing solutions (calcium paradox). Cardiovascular Research 1, 201-209(1967). Addendum Nayler and [46,242-249 propranolol
co-workers in a recent publication (1980)] have described the protective on the reperfused myocardium.
of the American Journal of Cardiology effect of verapamil, nifedpine and
Effect of a Calcium Channel on the Ischemic-Reperfused E. WEISHAAR
AND RICHARD
Blocker, Heart
J. BING
Institute of Applied Medical Research and Huntington Memorial Hospital, 100 Congress Street; Pasadena, Galajknia 91105, and the California Institute of Tec~n~~~~, Division of Chemistry and Chemical Eng~~er~ng, Pasadena, ~a~~~ar~~~, U.S.A.
Huntington
(Received 15 Afovember
1979; accepted >n revisedform
25 March
1980)
R. E. WIXSHAAR AND R. J. BING. The Beneficial Effect of a Calcium Channel Blocker, Diltiazem, on the Ischemic-Reperfused Heart. Journal of Molecular and Cellular Cardi&%s (1980) 12,993-1009. Diltiazem-HCl,@ a potent calcium channel blocker, was found to be effective in moderating the harmful effects of reperfusion on the severely ischemic myocardium. In isolated working rat heart preparations it was found that 120 min of giobal &hernia followed by 15 min of reperfusion resulted in a massive leakage of creatine phosphokinase into the coronary effluent, and in many cases, in fibrillation and/or contracture. Left ventricular end-diastolic pressure increased sharply during reperfusion in these hearts. Reperfusion did not affect tissue ATP levels, but did increase creatine phosphate somewhat. When Diltiazem was added to the per&sate (final concentration 0.4 PM) 5 min prior to re-establishment of flow, the deleterious effects of reperfusion were greatly reduced. None of the hearts fibrillated on reperfusion, and none developed contracture. Left-ventricular end-diastolic pressure was increased only slightly in these hearts. The amount of creatine phosphokinase released into the coronary effluent during m-flow was only one-half that which was released by hearts reperfused in the absence of Diltiazem. In the Diltiazem-treated hearts reperfusion restored creatine phosphate to near-normal levels, although ATP levels were not increased. The beneficial effects of Diltiazem are probably related to its ability to reduce the rapid and massive mitochondrial calcium overloading which normally accompanies reperfusion of severely ischemic myocardium.
KEY WORDS: Calcium channel blockers; Global ischemia; Reperfusion; Enzyme release; High energy phosphates; Left ventricular pressure.
Contracture;
1. Introduction The efficacy of reperfusing ischemic tissue is currently a controversial subject [4, 8, 23,291. Studies have shown that in a number of instances abrupt revascularization of the myocardium leads to a paradoxical extension of the damage due to &hernia alone [5, 6, 2.5, 401. The biochemical basis for this finding is not presently known, but is apparently related to the inability of the severely ischemic myocardium to maintain normal calcium homeostasis during reperfusion [14, 411. Shen and Jennings were the first to demonstrate that a sudden restoration of Aow Address for reprints: Richard J. Bing, M.D., Street, Pasadena, CA 91105, U.S.A. 0022~2828/80/ 100993+ 17 $02.00/O
Huntington
Memorial
0 1980 Academic
Hospital,
100 Congress
Press Inc. (London)
Limited
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R. E. WEISHAAR
AND
R. J. BING
leads to a rapid and massive influx of calcium ions into the ischemic region, which are subsequently accumulated within the mitochondrial matrix and precipitated as calcium phosphate [36]. This influx coincides with a large release of creatine phosphokinase from the myocardium and is, according to Jennings, responsible for the development of contracture bands in the ischemic-reperfused region
[201. It therefore seemed appropriate to examine the effectiveness of a new calcium channel blocker,* Diltiazem-HCl, in reducing reperfusion-induced damage to the ischemic myocardium. The precise mechanism by which Diltiazem alters calcium influx is unclear. Saikawa et al. have indicated that Diltiazem acts as an inhibitor of the transmembrane “slow channel” through which calcium ions enter the cell [33]. However, other recent studies on the effect of Diltiazem on smooth muscle have suggested that Diltiazem might act instead through inhibition of the regenerative release of activator calcium from the internal stores [X, 271. Diltiazem has also been shown previously by Weishaar et al. to diminish the rate of ATP hydrolysis and to decrease fatty acid and lactate levels in the ischemic myocardium [39]. Nagao and co-workers also found that Diltiazem administration prevented the depression of state 3 respiration in mitochondria normally observed following brief periods of ischemia [27]. In the present study the effects of reperfusion following a lengthy period of ischemia were examined using isolated, working rat hearts, in which the degree of flow reduction and other experimental conditions could be closely controlled. The results demonstrate that Diltiazem, when present in the circulating perfusate, significantly reduced the deleterious effects of reperfusion on the ischemic myocardium. 2. Materials
and Methods
Exferimental
model
Male Sprague-Dawley rats, weighing 250 to 400 g each and fasted overnight prior to the experiment, were used throughout the study. The animals were i.p.) and then injected intraanesthetized with Nembutal (40 mg/ animal, venously with heparin (2.5 mg). Ten minutes after the administration of heparin the heart was rapidly excised and placed in a beaker of ice-cold saline until contractions ceased (20 to 30 s). The hearts were perfused according to the working heart model described by Neely and Rovetto [29]. The entire system was water-jacketed and the temperature maintained at 37°C. After cannulating the aorta a preliminary washout is a more suitable term than “calcium “calcium channel blocker” * According to Katz, antagonist” since such agents are “known to ‘antagonize’ Ca2+ only at specific sites in the sarcolemma (the slow channel) and are virtually without effect on a variety of Ca2+-binding and transport systems” 1211.
CALClUM
CIIANNEL
BLOCKERS
ANI)
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perfusion was begun from a reservoir located 60 cm above the heart. The pcrfusate consisted of Krebs-Hens&it buffer containing 11 mu glucose and 10 mg;‘l heparin, and was bubbled xvith 1)5?; 0, : S?; (D,. (Heparin bvas nor present in the medium during the subsequent working perfusion.) ‘l’hc heart was perfused in this manner for 10 min, during kvhich time the left atrium \cas cannulated via the pulmonary veins. Once the atrium was cannulated, flo\~ from the ~~ashout reservoir wxs stopped and the heart immediately placed in a working mode ivith the left atria1 Ming pressure set at 10 cm H,O and the afterload at 60 mmHg hydrostatic pressure. The perfusate { 130 ml) was thereafter continuously recirculated, although at 30 min intervals 50 ml were removed and an equal amount of f&h perfilsate MXS added to the oxygenation chamber of the apparatus. The ticarLs \vcrc electricall) paced at 225 to 275 beats/min, with the pacing voltage set at roughly lo”,, above the threshold level. ‘1%~ hearts were pcrfilsed in this fashion I‘or an additional 10 min before ischemia was begun. During this pre-ischcmic period hernodynamic measurements lvcrc made scvcral times to ensure the stability of the preparation before comrncncing the expcrimcnt. (:oronary flow was reduced by the placement of a one-\yay valve in the aortic outflow tract which effectively prevented retrograde perfusion of the coronary arteries during diastole (for a detailed drawing of the one-\vay valve see Neely and Iiovctto 1291). During the prc-ischemic and also the reperfusion phases of the cxpcrirncnt, coronary flow was diverted around this one-way valve by means of a bypass tube. Ischemia was induced by clamping the bypass tube and f‘orcing the aortic outflow to pass through the one-way valve. Following this maneuver vcntricular pressure dcvclopmcnt quickly began to decline, \vith peak systolic pressure being reduced by roughly 80’1~ of the pre-ischemic Ici~cl lvithin 10 min of the onset of ischcmia. Coronary flow declined by 80 to 900,,, during this period and thercafter fell to roughly 5T.b of the normal level. Low-flow ischcmia \vas maintained for a total of 120 min, at which time lhe hearts were reperfuscd for an additional 15 min (a number of hearts were not rcperfuscrl, and following 15, 30, 60, I20 or 135 min of ischcmia alone the experiment was terminated). A 2 h period of ischernia was chosen in the present study since preliminary experiments rcvealcd rhat reperfitsion following shorter periods of flow reduction ( 15 to 60 rnin) occasionally reversed the hcmodynamic and biochemical changes produced by ischernia, and rarely induced contracturc. Occasionally contractile activity ceased after 15 to 20 min of ischemia, or during the first minutes of reperf’usion. In most of these hearts this cessation could be rcvcrsed 1)~ increasing the pacing voltage slightly. I Io\\.ever, in a number of rtlc reperfused hearts cvcn extremely high pacing voltages could not elicit a contractilc response. In those hearts in which the effect of Diltiazem on reperfusion-induced damagewas studied, a 5 ml aliquot of fresh perfusate, in which an appropriate amount 01‘
996
R. E. WEISHAAR
AND
R. J. BING
Diltiazem had been dissolved to provide a circulating concentration of 0.4 mM, was added to the oxygenating chamber of the perfusion apparatus 5 min prior to the onset of reflow. In all other experiments an equal amount of fresh perfusate alone was added at this time. Preliminary experiments in this laboratory have shown that at the concentration used in the present study, Diltiazem exerts a negative inotropic and chronotropic effect on the perfused nonischemic rat heart. In unpaced hearts, the heart rate was reduced by 40 to 60% within 3 min of the addition of Diltiazem to the perfusate, and in the paced heart left ventricle systolic pressure (LVSP) and left ventricular dP/dt,,, were reduced by 20 to 30% of normal levels. These effects were not permanent, however, and a partial restoration of myocardial contractility began approximately 10 to 15 min after Diltiazem administration. Hemodynamic measurements Aortic and atria1 pressures were monitored by conne&ng pressure transducers (Gould No. P23ID) to side-arms on the respective ccannulae. In the ischemic preparations, the aortic pressure was measured below the one-way valve. Intraventricular pressures were measured by inserting a 20-gauge needle, fluid-filled and connected to a pressure transducer, through the apex of the heart and into the left ventricle. All three transducers were,in turn connected to an Electronics for -flow was estimated by collecting the Medicine Monitoring system. Coronary effluent from the pulmonary artery in a graduated cylinder. Measurements of coronary flow were made every 5 min during the first 30 min of ischemia, and at 30 min intervals thereafter. Biochemical analysis In a number of experiments samples of the coronary effluent (0.2 ml) were collected in cooled glass vials at the following times: prior to the onset of ischemia, after 60 and 120 min ischemia, and after 1, 3, 5, 10 and 15 min reperfusion. These were then quickly frozen until analysis. (In experiments where the effects of ischemia alone were monitored, these latter five samples were collected following 121, 123, 125, 130 and 135 min ischemia). Creatine phosphokinase (CPK) (ATP-phosphotransferase) activity in these samples of effluent was determined by the method of Rokalski [32]. The rate of CPK leakage was then expressed in international units per minute per gram dry weight according to the method described by Ganote and co-workers [PI. The total amount of CPK released during the 15 min period of reperfusion (or during the corresponding period of ischemia) was determined by Simpson’s rule [Z]. In hearts in which high energy phosphate levels were determined the left ventricle .was, at the indicated times, rapidly frozen using stainless steel tongs precooled for several minutes in liquid nitrogen. The samples were then wrapped in
CALCIUM
CHANNEL
BLOCKERS
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997
aluminium foil and stored in liquid nitrogen until they could be analyzed. The levels of the following high energy phosphates were measured in a portion of the frozen ventricle : creatine phosphate (CP), adenosine-5’-triphosphate (ATP), and adenosine-5’-monophosphate (AMP). adenosine-5~-diphosphate (ADP) These levels were determined in perchloric acid extracts of the tissue samples according to standard procedures employing NADH-coupled reactions [3]. The remaining portion of the left ventricle was used to determine the dry weight of the heart. In the absence of ischemia, these preparations typically maintained stable function of periods of 2 h or longer, as determined both metabolically and hemodynamically. Coronary flow remained above 10 to 12 ml/min, and ATP and CP levels were only 9.6% and 8.3%, respectively, below zero-time levels after 2 h of perfusion (data not shown). Reagents The reagents used in these experiments were of an analytical grade quality. Solutions were made in all-glass distilled water. Diltiaz~m-HCl~ was kindly provided by Tanabe Seiaku Co., Ltd., Osaka, Japan.
Statistical analysis Data were evaluated using Student’s t test, or when necessary, an “a priori” t test, and the results were expressed as the mean & the standard error 1371. In the case where total CPK release during the period of reperfusion, or the corresponding period of ischemia, was evaluated, the results were expressed as the mean i the 95% confidence limits, and the significance was ascertained by both a standard two-way Analysis of Variance and a one-way Analysis of Variance after first transforming the data to its logarithmic equivalent [Z, 371.
3. Results The consequences of reperfusion on the severely ischemic myodardium, in both the presence and the absence of Diltiazem, will be divided into hemodynamic and the metabolic effects. Efects of reperfission oa myocardial Aemodynamics As can be seen in Figure 1, the decline in coronary flow during the 2 h period of global ischemia was rapid, falling to a level which was only 5% of the pre-ischemic value. Reperfusion only partially restored coronary Aow to a normal level. Coronary flow was, however, significantly higher (P < 0.05) in the Diltiazem
998
R.
E.
WEISHAAR
AND
Time
R. J.
BING
(mm)
FIGURE 1. Coronary flow in ischemic-only hearts (O---O), and in hearts reperfused in either the presence (m--m) or absence (A-A) of Diltiazem. Hearts were perfused in a working fashion with Krebs-Henseleit buffer containing 11 rn~ glucose at 37°C. Changes in flow are expressed as a percentage of the pre-ischemic level. Each symbol represents the mean & S.E. of at least seven experiments. * Significantly (P < 0.05) .mcreased as compared to percentage change in corononary flow following a corresponding period of &hernia; 7 significantly (P < 0.05) increased as compared to percentage change in coronary flow in hearts reperfused in the absence of Diltiazem.
treated hearts than in the hearts reperfused in the absence of the drug, indicating that Diltiazem had a vasodilating effect on the coronary arteries (Figure 1). Global ischemia also produced a prompt decline in the left ventricular systolic pressure (LVSP), and a corresponding increase in left ventricular end-diastolic pressure (LVEDP) [Figure 2(a) and (b)]. Th ese changes reached their maximum after approximately 15 min of ischemia and remained relatively constant thereafter. Aortic and atria1 pressures also diminished during the period of ischemia. As can be seen in Figure 2(a), reperfusion in the absence of Diltiazem led to a resulting in many cases from the marked increase in end-diastolic pressure, development of contracture. In these hearts LVEDP more than doubled within the first few minutes of reperfusion and remained at this elevated level throughout the period of reflow. Systolic pressure increased during reperfusion as well [Figure 2(b)]. This increase was also a reflection of contracture development and did not represent a reversal of the effects of ischemia on myocardial contractile function. In the Diltiazem-treated hearts these deleterious effects of reperfusion onhemodynamic function were virtually eliminated [Figure 2(a) and (b)]. LVEDP did not rise during the period of reperfusion and, in some cases, was restored to
I
1000
R.
E.
WEISHAAR
AND
R.
J.
BING
near the pre-ischemic level. LVSP rose slightly during reperfusion, though the increase differed from that which was seen in the non-Diltiazem hearts in that the increase was not related to the development of contracture. The effects of ischemia and reperfusion on the incidence of contracture development are described in Table 1 (a) and depicted schematically in Figure 3. In the present study contracture was defined as the absence of contractile activity (systolic pressure = end-diastolic pressure) accompanied by a rise in the enddiastolic pressure of at least 150% of the pre-ischemic level. This definition is equivalent to that used to define contracture clinically [23, 421. Contractile activity frequently ceased on reflow, occasionally even in the Diltiazem-treated hearts, though the cessation could often be reversed by increasing the pacing voltage. Hearts in which contractile activity could be restored in this way were not designated as having developed contracture. As can be seen in Table 1 (a), none of the hearts in which Diltiazem was added to the circulating perfusate developed contracture during reperfusion, while 37.5% (six of 16) of the hearts reperfused in the absence of Diltiazem eventually went into a state of contracture. These latter hearts also experienced a much greater incidence of fibrillation during reflow. In Figure 3 tracings obtained from four representative experiments are shown. In two of the experiments Diltiazem was added to the perfusate and in two it was not. It can be seen that reflow in the absence of Diltiazem resulted in rapid contracture in one experiment, and in the other in a slow deterioration of contractile function leading ultimately to the development of contracture. In the Diltiazem-treated hearts reperfusion was found to have little if any effect on contractile function in one experiment, and in the other resulted in a partial reversal of the effects of ischemia on contractility. In the latter experiment LVEDP was eventually restored to near the pre-ischemic level and LVSP to approximately 50% of normal (Figure 3).
Effects
of
reperfusion on myocardial metabolism
Enzyme release Figure 4 illustrates the effects of ischemia and of ischemia followed by reperfusion on creatine phosphokinase (CPK) leakage into the coronary effiuent. Ganote has previously shown that enzyme release by the myocardium is most likely related to and a number of studies have shown increased cell membrane permeability [II], marked ultrastructural damage accompanying the release of CPK [9, 13, 221. In the present study only small amounts of CPK were released by the myocardium during the period of global ischemia (Figure 4). However, reperfusion, in the absence of Diltiazem, was found to trigger a sudden, massive release of CPK, (g dry wt)-i following 10 min of which reached a peak of 14.3 & 2.7 iu mini reflow. Even after 15 min of reperfusion considerable quantities of CPK were still being released by the non-Diltiazem hearts (12.5 f 2.6 iu min-l (g dry wt)-r)
+ 103.4 f
f
-86.0
limits
95% confidence
37.5%
144.1*?
f
4 6.0%
(77.9 to 266.6)
+205.1
-63.5
37.5%
6
16
120 min ischemia 15 min reperfusion
release
*
5.5%
(29.7 to 165.5)
70.1”
+ 118.4 & 22.2%
-71.5
0
0
12
120 min ischemia 15 min reperfusion ( + Diltiazem)
(P < 0.05) increased as compared to the total amount of CPK released during the corresponding period of &hernia. (P < 0.05) increased as compared to the total amount of CPK released from hearts reperfused in the presence of Diltiazem.
2.2 (1.1 to 4.2)
(g dry wt)-r (15 min (or final 15 min
7.6%
0.5%
alone
and CPK
(b) CPK release iu CPK released reperfusion)-r ischemia)
of of
End-diastolic pressure (percentage pre-ischemic level at conclusion experiment)
of
0
0
13
Ischemia
development
Systolic pressure (percentage of pre-ischemic level at conclusion experiment)
contracture
contracture
on contracture
of hearts developing
Number
Percentage
of hearts examined
of hearts developing
Number
(a) Contractwe development
1. The effect of reperfusion
* Significantly t Significantly
TABLE
1002
R. E. WEISHAAR Im reflow
120m ischemia
2m reflow
AND 3m reflow
R. J. SING 4m reflow
5m reflow
IO m reflow
15m reflow
63 ss Z? Oi
-
FIGURE 3. Pressure tracings from four representative experiments. The hearts were perfused as described in Figure 1, In the lower two experiments a 5 ml aliquot of fresh pet-I&ate, into which an appropriate amount of Diltiazem had been dissolved to provide a final concentration of 0.4 nlM, was added to the circulating perfusate 5 min prior to the onset of reperfusion. In the upper two experiments an equal amount of fresh pet-f&ate alone was added. The tracings indicate the level of left ventricular pressure following 120 min of ischcmia, and 1,2,3,4,5, 10 and 15 min of reperfusion.
(Figure 4). The total amount of CPK released during the 15 min period of reperfusion averaged 144.4 IV/g dry wt, as compared to an average of2.2 W/g dry wt during the corresponding period of global ischemia [Table 1 (b)]. Diltiazem did not entirely prevent the reperfusion-induced release of CPK by the myocardium, although its presence in the perfusate during reflow did contribute to a significant reduction (P < 0.05) in the total amount of enzyme released, and therefore presumably to a decrease in membrane and ultrastructural damage. As can be seen in Figure 4 and Table 1 (b), the total amount of enzyme released from the myocardium was only about half of that which was released by the hearts which were not treated with Diltiazem (70.1 iu (15 min)--r (g dry wt)p’ vs. 144.1 iu (15 min)-1 (gm dry wt)-r) [Table l(b)].
Energy metabolism Global ischemia led to a prompt reduction in tissue levels of ATP and creatine phosphate (CP), while at the same time increasing, transiently, the level of AMP
CALCIUM
CHANNEL
BLOCKERS
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REPERFUSION
1003
Time hn)
FIGURE 4. Myocardial enzyme release from ischemic only hearts ((II-O), and from ischemic hearts reperfused in either the presence (a-m) or absence (A-----A) of Diltiazem. Hearts were perfused as described in Figure 1, and samples of coronary effluent were taken at the following times: after the 10 min non-ischemic control period, after 60 and 120 min of &hernia, and after 1, 3, 5, 10 and 15 min of reperfusion (or after the corresponding period of &hernia). Each symbol represents the mean + LE. of five to seven experiments. The total amount of enzyme released during reperfusion was found to be significantly lower when .Diltiazem was included in the perfusate during reperfusion than when it was not, according to both methods of statistical analysis described in the text.
5(a) and (b)]. ADP levels remained fairly constant throughout the 120 min period of ischemia [Figure 5(b)]. In the absence of Diltiazem, reperfusion had little effect on myocardial energy stores. ADP did decrease significantly during reperfusion, but CP, ATP and AMP were not significantly altered [P’igure 5(a) and (b)]. Reperfusion in the presence of Diltiazem likewise had little if any effect on the level of ATP. However, reflow in the Diltiazem-treated hearts did produce a large and highly significant increase in the level of creatine phosphate from (5.5 j_: 1.7 pmol/g dry wt to 17.6 f 2.3 pmol/g dry wt) and a corresponding large decrease in AMP (from 5.7 pmol/g dry wt to 2.9 f 0.2 @mof/g dry wt). ADP was also significantly reduced in these hearts, but not to the degree which AMP was. It is of interest to note that the tissue level of ATP apparently had very little bearing on the development of contracture in the present study. As can be seen in Figure 5(a), ATP levels were essentially the same in hearts reperfused in either the presence or absence of Diltiazem (and for that matter in those hearts rendered ischemic for 30, 60 or 120 min), but only in those hearts in which Diltiazem was omitted from the perfusate during reperfusion did contracture occur (Table 1 (a)). [Figure
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135 Time (min)
FIGURE 5. The effect of ischemia plus reperfusion on (a) ATP (a---@) and creatine phosphate (A---A) levels, and on (b) ADP (a---m) and AMP (+---+) levels. Hearts were perfused as described in Figure 1, and were freeze-clamped in sitz~ at the following times: after the lo-min non-ischemic control period, after 15, 30, 60 or 120 min of ischemia, or after 120 min of ischemia and 15 min of reperlnsion. In some experiments Diltiazem was added to the circulating perfusate 5 min prior to the onset of reperfnsion (open symbols). Each symbol represents the mean f S.E. of six to 10 hearts. * Significantly (P < 0.05) increased [Figure 4(a)] or decreased [Figure 4(b)] as compared to level following 120 min of &hernia. t Significantly (P < 0.05) to level in hearts reperfused in the increased [Figure 4(a)] or decreased [Figure 4(b)] as compared absence of Diltiazem.
4. Discussion Jennings and his colleagues were the first to report, in 1960, that reperfusion of ischemic myocardial tissue is not necessarily beneficial to the heart, and may in fact have the paradoxical effect of exacerbating the existing damage [I??]. The biochemical basis for this phenomenon is not known at the present time, but is thought to be related to changes which occur in the normal regulatory processes of the heart during ischemia, such that during reperfusion the injured cells rapidly take up calcium and other ions, leading to cell swelling and/or rupture. This uptake of calcium ions either causes it or is accompanied by marked changes in the myocardial ultrastructure, including the formation of contracture bands and separation of the basement membrane, as well as leakage of myocardial enzymes into the interstitial fluid [IO, 16, 22, 431. At the present time information is scarce concerning therapy designed to prevent the often harmful effects of reperfusion on the severely ischemic myodardium, although Ashraf and Bloor have shown that reperfusion with calcium-free plasma prevented the damage to the myocardial ultrastructure that otherwise accompanied reflow [I], and a recent preliminary report indicated that during ischemic arrest, Diltiazem might provide better protection for the myocardium than potassium [38]. In addition, Reimer et al. found that the necrosis which normally
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accompanies reperfusion in dogs was reduced by pretreatment with another calcium channel blocker, verapamil [30]. The results of the present study demonstrate that inclusion of Diltiazem-HCI in the perfusate during reperfusion significantly reduced the deleterious effect of reflow in the heart. The protective effect was demonstrated in several ways, including (i) prevention of contracture, which frequently (37.5:4 of the time) occurred when ischemic hearts were reperfused in the absence of Diltiazem; (ii), a significant reduction in the amount of CPK release into the coronary effluent during reflow (even when corrected for the vasodilating effect of Diltiazem) ; and (iii) restoration of myocardial creatine phosphate to near-normal levels. Since tissue levels of calcium were not measured in hearts reperfused In either the presence or absence of Diltiazem, it is impossible to say for certain that the protective effect of Diltiazem was due to a diminished influx of calcium ions into the ischemic-reperfused region. However, in the Diltiazem-treated hearts reperfusion produced a much smaller release of CPK than in the non-Diltiazem treated hearts and therefore presumably a reduction in the amount of ultrastructural damage. Previous studies have shown that such damage is related to a massive influx of calcium ions into the cell [19, 201. In addition, preliminary experiments in this laboratory have shown that Diltiazem, at the level used in the present study, diminished myocardial contractile force in the non-ischemic perfused rat heart (LVSP and left ventricular dP/dt,,, being reduced by 10 to 25% of normal levels). One of the striking findings was that reperfusion in the Diltiazem-treated hearts produced a marked increase in the tissue level of creatine phosphate (CP being restored to nearly two-thirds of the pre-ischemic value), while at the same time having little effect on the level of ATP [Figure 5(a)]. Similar quantitative discrepancies between creatine phosphate and ATP recoveries following reperfusion of ischemic tissue have been reported in several other studies [7, 17, 3.5, 40). Both IsseIhard and Chong found that the rapid restoration of tissue creatine phosphate levels normally precedes a similar, though slower, restoration of ATP levels as well [7, 171. In 1970, Gudbjarnason proposed the existence of separate mitochondrial and myofibrillar compartments of ATP, with creatine phosphate acting as a “transport form” of high energy bonds between the two [12]. Since that time Saks and his co-workers have provided evidence supporting this concept [34]. In the light of these findings, the results of the present study may be interpreted as indicating that, in the hearts reperfused in the presence of Diltiazem, there is a rapid restoration of mitochondrial synthesis of creatine phosphate, although there apparently exists a block in either the transfer of CP to the myofibrils, or in the reconversion of CP to ATP in that compartment. It is not possible to say from the information presented whether the block is permanent or only temporary. As was mentioned previously, it is interesting to note that the tissue level ofATP 2s2
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had little bearing on the development of contracture in the present experiments. ATP levels were essentially the same (5 to 6 pmol/g dry wt) in hearts reperfused in either the presence or the absence of Diltiazem; however, only in those hearts in which Diltiazem was omitted from the perfusate during reflow did contracture occur [Table 1 (a)]. Therefore, it would appear that the development of contracture was more likely related to an excessive influx of calcium ions into the nonDiltiazem treated hearts, than to a diminution in the cellular level of ATP. Other studies have also attributed the onset of myocardial contracture to disturbances in calcium homeostasis [23, 421. These findings differ from a recent report by Hearse et at., who found that contracture in isolated ischemic rat hearts was initiated once the tissue level of ATP fell below 12 pmol/g dry wt [IS]. There are several differences between the two studies, including the manner in which the hearts were rendered ischemic and the fact that in Hearse’s study the hearts were not paced, and would therefore arrest soon after the onset of ischemia, thus maintaining higher levels of ATP than would be otherwise expected. In the present study hearts occasionally arrested after 15 to 20 min of ischemia (at which time the tissue levels of ATP would probably be near the level described by Hearse ed rsl. as being critical). However, increasing the pacing voltage by 10 to 20% immediately restored and maintained contractile function for the remainder of the i,schemic phase, during which time the level of ATP continued to decline. None of the hearts developed contracture during the ischemic phase of the experiment. The results of this study demonstrate the highly protective effect of Diltiazem This protective effect is presumably on the ischemic-reperfused myocardium. related to the ability of this compound to restrict the previously described massive influx of calcium ions into the ischemic myocardium during reperfusion. In the present experiments it could be seen that revascularization, when combined with Diltiazem pre-treatment, resulted in a partial reversal of the effects ofischemia on the myocardium. Though the present study was restricted to an examination of only Dihiazem, other calcium channel blockers, such as nifedipine or verapamil, might also have proven beneficial in reducing the effects of reperfusion on the severely ischemic myocardium. However, since the mechanisms by which these three compounds inhibit the movement of calcium vary somewhat, the validity of such a general statement must await further evaluation.
The authors are grateful assistance, and to Mrs manuscript. The research reported
to Dr Patrick Pinto and Dr Gunter Pawlik Enid Soares for her help in the preparation in
this
paper
was
supported
by
grants
for their of the from
The
CALCIUM OHANNEL BLOCKERSAND Margaret the
W.
Council
and for
Herbert
Tobacco
Hoover,
Jr,
Foundation,
Research-U.S.A.,
Inc.,
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Angeles,
York,
New
California
and
York.
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