Jpn. J. Pharmacol. 67, 225-232 (1995)

A Study on : I. Mechanism of Anti-ischemic Action of Dilazep Is Not Coronary but Decreased Cardiac Mechanical Function in the Isolated, Working Rat Hearts

A.N. Ehsanul Hoque, Nina Hoque, Hiroko Hashizume and Yasushi Abiko*

Department of Pharmacology, Asahikawa Medical College, Asahikawa 078, Japan

Received August 16, 1994 Accepted December 9, 1994

ABSTRACT-In the isolated, perfused working rat heart, ischemia (15 min) decreased the mechanical func- tion and the tissue levels of triphosphate and creatine phosphate and increased the levels of lac- tate and free fatty acids. Reperfusion (20 min) did not restore the mechanical function, but restored in- completely the levels of metabolites, with the exception of free fatty acids, which increased further during reperfusion. Dilazep was given 5 min before starting ischemia until the end of ischemia. Dilazep at 5 or 10 pM decreased the cardiac mechanical function, but did not affect coronary flow in the pre-ischemic heart. Dilazep at 5 or 10 pM accelerated the recovery of mechanical function and coronary flow during reperfu- sion, and it attenuated metabolic changes induced by ischemia and reperfusion. Dilazep at 1 pM neither decreased the pre-ischemic mechanical function nor restored the mechanical function during reperfusion, although it attenuated the accumulation of free fatty acids during reperfusion. These results suggest that dilazep attenuates both ischemia- and reperfusion-induced myocardial damage and that the anti-ischemic action of dilazep is not due to coronary vasodilation but probably due to an energy-sparing effect and other effects that remain to be studied.

Keywords: Dilazep, Working heart, Ischemia, , Free fatty acid

Dilazep inhibits uptake of adenosine by cardiac cells mia reduces cardiac mechanical function, decreases the and hence potentiates the action of adenosine to dilate the tissue levels of high-energy phosphates such as adenosine coronary artery (1), suggesting that it has an anti-ischemic triphosphate (ATP) and creatine phosphate (CrP), and action. In fact, the cardioprotective (or anti-ischemic) increases the tissue levels of lactate and free fatty acids effect of dilazep was reported in the dog heart (2-4). In (FFA) (7, 8). Post-ischemic reperfusion (simply expressed clinical studies, dilazep has been used successfully as a as reperfusion in the present study) returns the mechani- drug for treatment of patients with ischemic heart disease cal and metabolic levels toward their pre-ischemic levels, (5, 6). However, the coronary vasodilating action of except for the levels of FFA, which further increase dur- dilazep is not very marked, suggesting that there is ing reperfusion (8, 9). Therefore, reperfusion-induced another mechanism for the anti-ischemic action of damage was evaluated by the tissue levels of FFA, particu- dilazep. larly the ratio of the level of arachidonic acid in the In the present study, therefore we examined whether ischemic heart to that in the non-ischemic heart, and the dilazep has anti-ischemic action in concentrations that do ratio of the arachidonic acid level in the reperfused heart not increase coronary flow. To exclude extracardiac fac- to that in the ischemic heart, because arachidonic acid in- tors, we employed an isolated working rat heart prepara- creases markedly during both ischemia and reperfusion. tion. Ischemia-induced damage was evaluated by both mechanical and metabolic functions of the heart; ische- MATERIALS AND METHODS t Part of the present study has been reported at the 44th Regional Meeting (Kita Area) of The Japanese Pharmacological Society, Heart perfusion Hirosaki, October 15-16, 1993. Male Sprague-Dawley rats (280-340 g; Sankyo Labo *To whom correspondence should be addressed . Service Corporation, Sapporo) were anesthetized with so- dium pentobarbital (50 mg/kg, i.p.). After thoracotomy, Preparation of frozen cardiac tissue samples the hearts were quickly removed and then perfused ac- The heart was frozen with a Wollenberger's clamp previ- cording to the Langendorff method followed by the work- ously cooled with liquid nitrogen (-173 IC). The freeze- ing heart method. The solution for perfusion was a clamped cardiac tissue samples were stored in liquid nitro- modified Krebs-Henseleit bicarbonate (KHB) buffer gen until the biochemical analysis was performed. (119.4 mM NaCl, 4.7 mM KCI, 2.9 mM CaC12, 1.2 mM MgSO4i 1.2 mM KH2PO4, 25.0 mM NaHCO3, 11.0 MM Assay of the tissue high-energy phosphates and lactate glucose and 0.5 mM EDTA-2 Na) equilibrated with a gas A part of the frozen cardiac tissue sample (about mixture of 95010 02 + 5% CO2 and maintained at 371C. 0.8 -1.0 g) was pulverized in a mortar and pestle cooled Hearts were initially perfused using the Langendorff to the temperature of liquid nitrogen. ATP, adenosine method at a constant pressure of 90 cmH2O for 10 min diphosphate (ADP), (AMP), and then perfused using the working heart method at a CrP and lactate were extracted from the pulverized tissue left atrial filling pressure of 12.5 cmH2O and an afterload sample with perchloric acid, and then neutralized with pressure of 90 cmH2O for 15 min (8). Ischemia was in- KOH solution. These metabolites of energy metabolism duced by lowering the afterload pressure from 90 cmH2O were assayed by standard enzymatic methods (11 - 13) to 0 cmH2O (i.e., no-afterload ischemia) (10). Reperfusion using a spectrophotometer (Gilford System 2600; Gilford was induced by returning the afterload pressure to its ini- Instrument Laboratories, Inc., Oberlin, OH, USA). tial level (90 cmH2O). Aortic pressure and heart rate were Energy charge potential (ECP) was calculated according monitored with a pressure transducer placed in the aortic to the following formula: cannula. Cardiac mechanical function was defined as ECP = (ATP + 0.5 ADP)/(ATP + ADP + AMP) RPP (peak aortic pressure multiplied by heart rate). Coro- nary flow (defined as the flow coming from the cannula in- Assay of the tissue FFA serted into the pulmonary artery) was measured with a The levels of tissue FFA were measured according to graded glass cylinder. the method described in our previous report (8). Briefly, the frozen cardiac tissue (about 150 mg) was pulverized in Experimental protocol and heart groups a mortar cooled with liquid nitrogen, and tissue FFA were The hearts were divided into 4 groups: control (no extracted from the pulverized tissue with chloroform/ drug), 1 pM dilazep, 5 pM dilazep and 10 pM dilazep methanol (2:1) containing 0.05% butylated hydroxy- groups. In each group, the hearts were divided further toluene (as an anti-oxidant), and then the FFA in the into 3 subgroups: non-ischemia or pre-ischemia (designat- extract were converted to their fluorescent derivatives ed as NI), ischemia (designated as I), and reperfusion with 9-anthryldiazomethane in methanol. After incuba- (designated as R) subgroups. In our previous study, we tion at room temperature for 1 hr, the fluorescent-deriva- found that 15 min of ischemia is sufficient to produce ir- tives of FFA were filtered with a Millipore' filter (FH reversible damage to the heart in terms of mechanical and 0.5 pm; Nihon Millipore Kogyo K.K., Yonezawa) and metabolic damage. Therefore in the present study, ische- injected into a reverse-phase high-performance liquid mia was performed for 15 min, and reperfusion was start- chromatography system with a Zorbax-ODS column ed immediately after ischemia and continued for 20 min. (0.46 x 25 cm; DuPont, Philadelphia, PA, USA). In NI, I and R groups, the hearts were freeze-clamped im- Methanol/distilled water (100:80) was used as the mobile mediately before starting ischemia, immediately after the phase. The level of individual FFA was determined by end of ischemia, and immediately after the end of reperfu- comparing the peak height of the FFA with that of a sion, respectively. In the drug-treated groups, dilazep (1 known amount of heptadecanoic acid (an internal stand- pM, 5 pM or 10 pM) was administered to the perfusion ard). solution 5 min before starting ischemia until the end of ischemia, and then reperfusion was performed with nor- Drugs mal KHB buffer that did not contain dilazep (Fig. 1). Dilazep hydrochloride was kindly supplied by Kowa Therefore, there were the following 12 groups: control: Company (Tokyo). The drug was dissolved in the normal NI (n = 10), control:I (n = 10), control: R (n = 12); 1 pM KHB buffer used in the present study. Biochemicals, re- dilazep:NI (n=6), 1 pM dilazep:I (n=7), 1 pM dilazep:R agents, and were purchased from Sigma Chemical (n=8), 5 pM dilazep:NI (n=7), 5 pM dilazep:I (n=7), Company (St. Louis, MO, USA). 5 pM dilazep:R (n=7), 10 pM dilazep:NI (n=6), 10 ttM dilazep : I (n = 7), 10 pM dilazep: R (n = 7). Statistical analyses All data are expressed as means±S.E.M. The signifi- cance of difference between means was analyzed by the analysis of variance, followed by Duncan's multiple- ml/min (upper panel), decreased the heart rate to 0/min range test for unpaired observations and by Student's t- and also decreased aortic pressure to 0 mmHg; therefore, test for paired observations. A P value of 0.05 or less was RPP became 0 mmHg/min after ischemia (lower panel). considered significant. After reperfusion following ischemia, coronary flow re- covered, although incompletely, but RPP that had been RESULTS decreased by ischemia did not recover, indicating that ischemia and reperfusion produced mechanical dysfunc- Coronary flow and RPP tion of the heart. Changes in coronary flow and those in RPP during In the pre-ischemic (i.e., non-ischemic) heart, dilazep non-ischemia, ischemia and reperfusion in the presence or at 1 jM, 5 uM or 10 uM did not affect coronary flow sig- absence of dilazep are illustrated in Fig. 1. A change of nificantly (upper panel, from 10 to 15 min), but it perfusion from the Langendorff to the working heart decreased RPP in a concentration-dependent manner method increased coronary flow and RPP. In the control (lower panel, from 10 to 15 min). The decrease in RPP of experiments, ischemia decreased coronary flow to 0 the pre-ischemic heart induced by dilazep at 5 pM or 10 pM was significant statistically. Even in the presence of dilazep, ischemia decreased both coronary flow and RPP. The dilazep-treated heart was then reperfused. After 20 min of reperfusion of the ischemic heart treated with dilazep at 5 pM or 10 pM, there was a significant degree of recovery in both coronary flow and RPP, indicating that dilazep at 5 pM or 10 pM restored coronary flow and mechanical function during reperfusion following ische- mia toward the pre-ische-mic level. Dilazep at 1 pM, which did not significantly change RPP in the pre-ische- mic heart, however, did not accelerate the recovery of either coronary flow or RPP during reperfusion following ischemia. Because dilazep in the concentrations used in the present study did not have a coronary dilating effect in the non-ischemic heart, the dilazep-induced increase in coro- nary flow during reperfusion is regarded as a phenom- enon secondary to an increase in the cardiac mechanical function induced by the drug, which increases coronary venous drainage, hence coronary flow.

High-energy phosphates In Fig. 2, changes in the levels of both ATP (upper panel) and CrP (lower panel) during non-ischemia, ische- mia and reperfusion in the presence or absence of dilazep at 1 pM, 5 pM or 10 pM are shown. In the control group, the tissue level of ATP markedly decreased after ische- Fig. 1. Effects of dilazep at 1 pM, 5 pM and 10 pM on the coro- mia, but it recovered after reperfusion, although incom- nary flow (upper panel) and RPP (lower panel). After the preliminary perfusion for 10 min (from 0 to 10 min), the perfusion pletely; the level of ATP in the control:I group was sig- was continued for 5 min with either normal KHB buffer or solution nificantly lower than that in the control:N1 group and containing 1 uM, 5 pM or 10 IM dilazep. Drugs were administered also significantly lower than that in the control:R group. 5 min before ischemia until the end of ischemia. In the control Dilazep at 1 uM, 5 pM or 10 pM did not affect the basal group, drugs were not administered. All data are expressed as means±S.E.M. Control ((D), control:R group (n=12); 1 ItM level of ATP in the non-ischemic heart. The levels of ATP dilazep (40), 1 1iM dilazep:R group (n=8); 5 pM dilazep (A), 5 pM in the 10 pM dilazep:I and 5 pM dilazep: groups, dilazep:R group (n=7); 101iM dilazep (A), 10 IM dilazep:R group however, were significantly higher than that in the con- (n = 7). *P <0.05, compared with the corresponding value in the con- trol:I group. The levels of ATP in the 10 pM dilazep:R trol group. For example, in the lower panel, the value of the RPP of 5 pM dilazep:R or 10 1,M dilazep:R group at 40 min (10 min after and 5 pM dilazep:R groups were also significantly higher reperfusion) is significantly higher than that in the control:R group than that in the control:R group. at 40 min. The level of CrP in the control group decreased marked- ly after ischemia and recovered completely after reperfu- sion significantly. Higher levels of CrP induced by dilazep sion. Dilazep at 10 pM increased the basal level of CrP in before ischemia are probably due to a decrease in cardiac the non-ischemic heart. Dilazep at 10 pM or 5 pM at- mechanical function induced by the drug. The CrP levels tenuated the decrease in CrP caused by ischemia sig- in the 5 pM dilazep:I or 10 ,uM dilazep:l group were sig- nificantly, and it increased the level of CrP after reperfu- nificantly higher than that in the control:l group, and the UP levels in the 5 [tM dilazep:R and 10 pM dilazep:R groups were also significantly higher than that in the con- trol:R group. Changes in ECP are shown in the upper panel of Fig. 3. The results with ECP were essentially the same as those with ATP.

Lactate Changes in the level of lactate during ischemia and reperfusion are shown in the lower panel of Fig. 3. In the control group, the level of lactate increased significantly after ischemia compared to the non-ischemic group and

LJIIQLG'.J L_"1IQLG`..J L.,1IQLGP Fig. 2. The levels of ATP (upper panel) and CrP (lower panel) in the rat heart. In the NI (non-ischemia), I (ischemia) and R (reperfu- sion) groups, the heart was freeze-clamped immediately before ischemia, at the end of ischemia and at the end of reperfusion, respectively. See the text for names of the groups (Materials and Methods). Drugs were given from 5 min before ischemia to the end of ischemia. Each group consists of more than 6 hearts. All data are expressed as means±S.E.M. tP <0.05, between two subgroups in the control, 1 pM dilazep, 5 pM dilazep or 10 1,,M dilazep group. Fig. 3. The tissue levels of ECP (upper panel) and lactate (lower *P<0 .05, compared with the value in the corresponding subgroup panel) in the rat heart in Fig. 2. Symbols are the same as those in (NI, I or R) in the control group. Fig. 2. decreased markedly after reperfusion compared to the and palmitoleic acids was more markedly attenuated by ischemic group. The lactate level in the 10 pM dilazep:I dilazep during reperfusion than that during ischemia. In group was significantly lower than that in the control:I Fig. 5, changes in the levels of linoleic (upper panel) and group. The level of lactate in the 10 pM dilazep:R or 5 stearic (lower panel) acids are illustrated. In the control pM dilazep:R group was significantly lower than that of group, these FFA increased after ischemia and further in- the control:R group. creased markedly after reperfusion. The effect of dilazep on the tissue levels of linoleic and stearic acids was essen- Arachidonic acid and other FFA tially the same as that on the levels of arachidonic and In Fig. 4, changes in the levels of arachidonic (upper palmitoleic acids; dilazep at 10 pM significantly attenuat- panel) and palmitoleic (lower panel) acids during ische- ed the accumulation of stearic acid during ischemia, and mia and reperfusion are illustrated. In the control group, dilazep at 1 pM, 5 pM or 10 uM significantly attenuated the levels of arachidonic and palmitoleic acids increased the accumulation of both linoleic and stearic acids during after ischemia, and further increased markedly after reper- reperfusion. fusion. The level of arachidonic acid in the 10 pM dilazep:I group was significantly lower than that in the control:I group, and the arachidonic acid levels in the 1 pM dilazep:R, 5 pM dilazep:R and 10 pM dilazep:R groups were significantly lower than that in the control:R group. A similar result was obtained with palmitoleic acid. It should be noted that accumulation of arachidonic

Fig. 4. The tissue levels of arachidonic acid (upper panel) and Fig. 5. The levels of linoleic acid (upper panel) and stearic acid palmitoleic acid (lower panel) in the rat heart in Fig. 2. Symbols are (lower panel) in the rat heart in Fig. 2. Symbols are the same as those the same as those in Fig. 2. in Fig. 2. DISCUSSION damages were attenuated by dilazep. The anti-ischemic effect of dilazep is possibly due to a decrease in cardiac The results of the present study clearly demonstrate mechanical function, because dilazep decreased pre- that dilazep has an anti-ischemic action in concentrations ischemic cardiac mechanical function concentration-de- that do not increase coronary flow of the pre-ischemic pendently and hence conserved the ATP level after ische- heart. Both ischemia-induced and reperfusion-induced mia. The degree of recovery of post-ischemic mechanical function was shown to depend on the level of ATP after ischemia (Fig. 6). Therefore, the anti-ischemic effect of dilazep on the ischemic heart can be interpreted as an energy-sparing effect (14). Recovery of coronary flow dur- ing reperfusion induced by dilazep at 5 pM or 101iM is probably due to recovery of mechanical function as men- tioned before. What is the mechanism of the decrease in cardiac mechanical function induced by dilazep? According to Tamura et al. (15), dilazep has a local anesthetic action, the extent of which is comparable to that of lidocaine. The fact that dilazep inhibits myocardial hypercontrac- ture induced by veratridine (16), which slows the inactiva- tion process of the Na+ channel, supports the view that the protective effect of dilazep is due to its Na+ channel blocking effect, leading to a decrease in mechanical func- tion of the heart. There are some interesting reports show- ing that dilazep produces a Ca" antagonizing effect in the guinea pig taenia coli (17) and in dog coronary artery (18). Dilazep also inhibits the histamine-stimulated cyto- solic Ca" increase in cultured endothelial cells (19). In the heart, both ischemia and reperfusion produce release of arachidonic acid and other FFA in the myocar- dium (8, 20, 21). This is because ischemia and reperfusion increase the intracellular concentration, leading to increased activity of the phospholipases and hence to increased release of FFA from phospholipids of the cell membrane (22-24). Anti-ischemic drugs such as j3- adrenoceptor antagonists and calcium channel blockers have been reported to attenuate the ischemia-reperfusion- induced FFA accumulation during both ischemia and reperfusion (8, 25). In addition, d-propranolol, which has Fig. 6. The relationship between the tissue levels of ATP and the practically no i3-adrenoceptor antagonistic action but has RPP values. Upper panel: Relationship between the decrease in a local anesthetic action, and lidocaine attenuate FFA ac- RPP before ischemia and the decrease in the ATP level induced by cumulation during ischemia and reperfusion (26, 27). ischemia, showing that the more RPP decreases before ischemia, the By what mechanism did dilazep attenuate the ischemia- smaller the ischemia-induced decrease in the ATP level is. Lower and reperfusion-induced FFA accumulation? FFA ac- panel: Relationship between the value of RPP at the end of reperfu- sion and the ATP level immediately after ischemia, showing that the cumulation is thought to be a result of increased deacyla- higher the ATP level after ischemia is, the higher the RPP level after tion and/or decreased reacylation of membrane phos- reperfusion is. In both the upper and lower panels, the data are those in Fig. 1 (lower panel) and Fig. 2 (upper panel), and only the mean pholipids (28). During ischemia, there was a decline in the values in the control, dilazep at 1 iM, 5 pM and 10 f1M groups are level of ATP that regulates the activity of acyl-CoA syn- plotted. Taking both data into account, a decrease in RPP thetase, which is a rate limiting factor of phospholipid (mechanical function) would minimize the decrease in the ATP level synthesis (29). This suggests that one of the major mecha- after ischemia, leading to a better recovery of mechanical function nisms of FFA accumulation during ischemia is a decrease after reperfusion. This is an example of the energy-sparing effect. Since dilazep decreases RPP before ischemia, it minimizes the in reacylation to phospholipids. Because dilazep attenu- decrease in the ATP level after ischemia, leading to a better recovery ates the decrease in the ATP level during ischemia, it in- of mechanical function. hibits FFA accumulation during ischemia. Deacylation of phospholipids should be accelerated by ischemia, because In conclusion, dilazep attenuates the ischemia-reperfu- there is an increased activity of phospholipase A2 during sion-induced damage of the heart probably because of its ischemia (30), which results from an increase in intracel- energy-sparing effect and some other effects that remain lular levels of Cat+. to be studied. The mechanism of FFA accumulation during reperfu- sion is not the same as that during ischemia, because fur- Acknowledgments The authors wish to express their sincere thanks to Mr. Tadahiko ther accumulation of FFA occurs during reperfusion even Yokoyama for his skillful technical assistance, and members of the when the ATP level increases. There is circumstantial evi- Department of Pharmacology for their kind advice and help in mak- dence that myocardial phospholipases A2 and C mainly ing this study. 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