Electronmicroscopic Autoradiographic Study of the Distribution of 3H-Diltiazem in Myocardial Cells

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1 Electronmicroscopic Autoradiographic Study of the Distribution of 3H-Diltiazem in Myocardial Cells Motoki TAGAMI, M.D., Yasuo NARA, Ph.D.,* Akiyoshi KUBOTA, M.D., Toshiaki SUNAGA, M.D.,** Hidenori MAEZAWA, M.D.,*** Ryoichi HORIE, M.D.,* and Yukio YAMORI, M.D.* SUMMARY The distribution of 3H-diltiazem in myocardial cell organelles was studied using an electronmicroscopic autoradiographic technique. At 3, 5 and 10min after 3H-diltiazem injection, silver grains, which indicate the existence of diltiazem, were detected in plasma membranes and intracellular organelles. Silver grains in Tsystems were observed more frequently at 3min than at 5 and 10min. In contrast, silver grains in sarcoplasmic reticula and mitochondria apparently increased with the passage of time. These findings suggest that diltiazem is transported through the T system and may accumulate within the sarcoplasmic reticula and mitochondria. Furthermore, our study revealed morphologically for the first time that the intracellular sites of action where diltiazem was directed were possibily the mitochondria. Additional Indexing Words: Calcium antagonist Autoradiographic analysis Circle method Sarcoplasmic reticulum Mitochondria T has been suggested that calcium antagonist drugs, in general, have a beneficial effect on experimental cardiac ischemia.1)-3) Recently, several groups have shown that some of the in vivo consequences of myocardial ischemia, particularly mitochondrial damage, can be attenuated by the pretreatment of the heart with calcium antagonists such as verapamil or diltiazem.4),5) In spite of intensive research, neither mechanisms of ischemic damage to mitochondria nor exact modes of action of these drugs are thus far well established. The purpose of this study was to clarify, using an elec- From the Department of Medicine, Sanraku Hospital, Chiyoda-ku, Tokyo,* Department of Pathology, Shimane Medical University, Izumo,** Department of Medicine, Saga Medical University, Saga, and *** Third Department of Medicine, Tokyo Medical and Dental University, Tokyo, Japan. Address for reprint: Motoki Tagami, M.D., Department of Internal Medicine, Sanraku Hospital, 2-5 Kanda-Surugadai, Chiyoda-ku, Tokyo 101, Japan. Received for publication December 4, Manuscript revised March 8,

2 824 TAGAMI, ET AL. Jpn. Heart J. September 1985 tronmicroscopic autoradiographic technique, whether or not diltiazem can pass through sarcolemma and distribute into myocardial cell organelles. METHODS Ten male Wistar-Kyoto rats (WKY) from 10 to 12 weeks of age were used. Eight rats were anesthetized with pentobarbital and then received injections via the right femoral vein of 1.0mCi/100g body weight of 3Hdiltiazem (Tanabe Pharmaceutical Co., Ltd.; specific activity 27.1mCi/mg). One mci of 3H-diltiazem was dissolved in 0.5ml of physiological saline. Two rats were injected with physiological saline alone as controls. The hearts of WKY were carefully removed 1, 3, 5 and 10min after the 3H-diltiazem injection and 5min after physiological saline injection. Afterwards the hearts were cut into small pieces which were fixed with the fixative containing 2.5% glutaraldehyde and 1% H2PtCl6 buffered with 0.1M cacodylate buffer for 3 h at 4 Ž, and then washed overnight at 4 Ž in 0.1M cacodylate buffer containing 1% H2PtCl6. After post-fixation with 2% OsO4 buffered with 0.1M cacodylate buffer for 2 h, tissue sections were dehydrated in graded ethanol and embedded in Epon 812. Thin sections, cut on a ultratome, were placed on slides coated with collodion. Individual slides were then coated with Sakura NR-H2 photographic emulsion diluted 1: 5, and were exposed for 4 to 6 weeks under refrigeration at 4 Ž. Following exposure, these slides were developed in Kodak Microdol-X. After development, the thin sections on the slides were transferred to electronmicroscopic grids by Kopriwa's method6) and were stained with lead citrate. The test conducted at the time of filming verified that the thickness of the emulsion over the slide was an even monolayer. The unlabeled tissue section slide revealed the presence of almost no silver grains; therefore, every grain observed in the labeled tissue was considered to be significant. A sampling of 80 to 100 micrographs, drawn in approximately equal number from longitudinal and transverse sections of the myocardium, was made from the autoradiographic preparations of each rat heart. Each micrograph contained at least one developed grain (average number of grains per 35 sq ƒêm field: 6.7) and was printed at a final magnification of 20,000 ~. A circle with a radius of 2.8mm (corresponding to 140nm) was drawn around each silver grain, centered on the midpoint of the long axis of the grain, as shown in Fig.1. Circles of this size have a 25% probability of enclosing the radioactive source and were used to determine the relative distribution of tritium-labeled diltiazem among the structural components of the myocardium.1),8) Any structures that were even partially enclosed by these

3 Vol.26 No.5 DISTRIBUTION OF DILTIAZEM IN MYOCARDIAL CELLS 825 circles were considered to be primary potential sites of labeled diltiazem molecules and were recorded as being associated with the grains. The percentage of grains thus associated with each of the myocardial and sarcoplasmic structures was then calculated. Five reference circles of the same size, representing a random sampling of enclosed tissue elements, were applied to each micrograph, one near the center and one near the center of each quadrant, by means of the grid illustrated in Fig.1. The percentage of these random circles associated with each tissue component provided a measure of the expected frequency of the appearance of structures within circles. These percentages were used for comparison with the distribution of structures found among the grain-associated circles.9),10) The radioactivity of tissue sections before and after fixation and dehydration was measured by a liquid scintillation counter (Tri-Carb 460 CD; Packard Co., Ltd.). RESULTS We examined the distribution of intravenously injected 3H-diltiazem in myocardial cells by using an electronmicroscopic autoradiographic technique. The data of 3H-diltiazem distribution were obtained from both longitudinal and transverse sections of the myocardial cells. At 1min after injection we were not able to detect sufficient silver grains to indicate 3H-diltiazem. The difficulty in detecting 3H-diltiazem at 1min may be related to the concentration of 3H-diltiazem in the cell organelles and also to the sensitivity of photographic emulsion. At 3, 5 and 10min after injection sufficient silver grains (average number of grains per 35 sq Đm field: 6.7) were observed. The circle method of analysis was utilized to determine the relative concentration of labeling in the different structural components. Table I-(1) shows the number of individual grains examined and the percentage of those grains associated with the various myocardial and sarcoplasmic structures at 3, 5 and 10min after injection. In order to obtain an appropriate basis for evaluating the relative distribution of autoradiographic grains on the myocardium, random circles were similarly assayed in the micrographs. The results shown in Table I-(2) represent the frequency with which myocardial and sarcoplasmic components were found to be associated with circles of the same size used for autoradiographic analysis but derived instead from a random sampling of myocardial space (Fig.1). Thus, these numbers indicate the percentage of

4 826 TAGAMI, ET AL. Jpn. S Heart J. eptember 1985 Table I. Autoradiographic Grains and Random Circles Associated with Myocardial and Sarcoplasmic Structures (a) All values represent the means }1 standard deviation calculated from the sample means of the individual animals. (b) Includes glycogen and ribosomes. (c) Includes plasma membrane and intercalated disks. grain-associated circles that would be expected to enclose parts of each of these structures if autoradiographic grains were distributed at random. Comparing the data in Table I-(1), representing observed values, with the data in Table I-(2), expected values, a chi-square test of the six sarcoplasmic components gives 9.49 (p <0.1) for the myocardial cells at 3min after injection, 9.99 (p <0.1) at 5min and (p <0.05) at 10min. Thus, 3H-diltiazem in the myocardial cells was not randomly distributed among the sarcoplasmic structures. Dividing the numbers in Table I-(1) by the corresponding numbers in Table I-(2) leads to normalized values for the distribution of autoradiographic grains found over myocardial and sarcoplasmic structures, as shown in Table II. If the distribution of radioactively labeled diltiazem had shown no selective localization, then the figures in Table II would all have been about equal. It has been reported that diltiazem has its binding sites on the plasma

5 Vol 26 No.5 DISTRIBUTION OF DILTIAZEM IN MYOCARDIAL CELLS 827 Fig.1. Transverse section of an experimental myocardium printed with a superimposed grid for autoradiographic analysis. Black circles, calculated to have a 25% probability of enclosing the radioactive source, are drawn around the midpoints of actual autoradiographic grains. The 5 white circles are used for the random assay of structures enclosed in areas of the same size. Silver grains are developed over mitochondria, sarcoplasmic reticula, T systems and myofibrils. Five min after 3H-diltiazem injection. Bar indicates 1ƒÊ ~20,000. Table II. Normalized Distribution of Autoradiographic Grains over Myocardial and Sarcoplasmic Structures membrane (sarcolemma).11),12) In this experiment, however, the plasma membrane showed a low concentration of radioactivity, although more was expected. In contrast, greater labeling was demonstrated in the sarco-

6 828 TAGAMI, ET AL. Jpn.Hea 1985 Fig.2. a. Low magnification autoradiograph of myocardium. Silver grains are developed over T systems (arrows), mitochondria and myofibrils. Three min after 3H-diltiazem injection. Bar indicates 1ƒÊ ~12,000. b. Higher magnification of the area indicated by the double arrows. Silver grains are observed in subsarcolemmal cisterns, that is T systems. ~18,000. Fig.3. Autoradiograph of myocardium at 5min after 3H-diltiazem injection. Silver grains are developed over mitochondria and myofibrils. Bar indicates 1ƒÊ ~24,500. plasmic reticula, mitochondria and T system. The normalized labeling values of the sarcoplasmic reticula and mitochondria at 10min after injection were 1.30 }0.19 and 1.41 }0.08, respectively. These values were apparently

7 Vol.26 DISTRIBUTION OF DILTIAZEM IN MYOCARDIAL CELLS 329 No.5 Fig.4. a. Autoradiograph of myocardium at 5min after 3H-diltiazem injection. Silver grains are observed over plasma membranes, mitochondria, sarcoplasmic reticula and myofibrils. Bar indicates 1ƒÊ ~15,000. b. Higher magnification of the area indicated by the arrow. Silver grains are developed over mitochondria. ~30,000. higher than the ones at 3min but not significant statistically. Furthermore, the normalized labeling values of the T system at 3, 5 and 10min were 2.01 } 0.31, 1.42 }0.30 and 1.40 }0.41, respectively. The value at 3min was high but not significant by Student's t-test (Figs.2, 3, 4). It has been suggested that diltiazem has a strong affinity for heavy metals, such as platinum, vanadium and copper. It is also known that heavy metals are firmly bound to radicals of tissue proteins. Therefore, heavy metals should be bound to both diltiazem and tissue proteins at the same time and may secure diltiazem to the organelles during fixation and dehydration. For these reasons, platinum, vanadium or copper were added to the fixative and dehydrant. We then measured the radioactivity value of the tissue sections at 3min after 3H-diltiazem injection after fixation and dehydration. The radioactivity values with platinum, vanadium and copper were as follows: platinum-5.37 ~107 DPM/g, vanadium-1.04 ~107 DPM/g, copper-6.26 ~106 DPM/g. As a control we measured the radioactivity value of the same tissue sections without the addition of heavy metals during fixation and dehydration. The value was 2.24 ~106 DPM/g. The radio-

8 830 TAGAMI, ET AL. Jpn. Heart J. S eptember 1985 Table III. Radioactivity before and after Fixation and Dehydration of the Myocardium at 1, 3, 5 and 10Min after 3H-diltiazem Injection activity value with platinum was the highest as compared with the other metals and in fact was 20 times higher than the control. Therefore platinum was consistently added to the fixative and dehydrant in this experiment. The radioactivity values before and after fixation and dehydration of the myocardium are shown in Table III. DISCUSSION It is essential in electronmicroscopic autoradiography to bind diltiazem to the target organelles and to secure it to the organelles during fixation and dehydration. It has been suggested that diltiazem has a strong affinity for heavy metals. It is also known that heavy metals are firmly bound to radicals of tissue proteins. Therefore, heavy metals should be bound to both diltiazem and tissue protein at the same time. In this experiment we measured the radioactivity value of tissue sections with platinum, vanadium or copper during fixation and dehydration. The radioactivity value with platinum was the highest as compared with the other metals and in fact was 20 times higher than the one without. Moreover it was demonstrated that the radioactivity value after fixation and dehydration with platinum was about 85% before fixation and dehydration (Table III). These findings indicate that platinum was bound to both diltiazem and tissue protein and secured diltiazem to the target organelles. A full interpretation of autoradiograms depends on a knowledge of the expected grain distribution around radioactive sources. This distribution depends both on geometric factors and on photographic ones. Salpeter et al8) analyzed grain distributions around a radioactive source (consisting of 3Hpolystyrene) and demonstrated that a circle with a radius of 140nm drawn around each silver grain had a 25% probability of enclosing the radioactive source. In this study we intravenously injected 1.0mCi (0.037mg)/100g body weight of 3H-diltiazem. This dosage is within the pharmacologic range. We examined the distribution of 3H-diltiazem in myocardial cells by an

9 Vol.26 No.5 DISTRIBUTION OF DILTIAZEM IN MYOCARDIAL CELLS 831 electronmicroscopic autoradiographic technique and then analyzed the autoradiograms using the circle method in order to determine the true distribution of the drug among the structural components. At 1min after injection, silver grains, which indicate the existence of the drug, were not detected in the myocardial cells. The difficulty in detecting the drug at 1min may be related to the concentration of the drug and also on the sensitivity of photographic emulsion. At 3, 5 and 10min after injection sufficient silver grains were detected in the myocardial cells. A chi-square test of the six sarcoplasmic components, comparing the data in Table I-(1), representing observed values, with the data in Table I-(2), expected values, gives 9.49 (p<0.1) for the myocardial cells at 3min after injection, 9.99 (p <0.1) at 5min and (p<0.05) at 10min. These results revealed that silver grains, 3H-diltiazem, in the myocardial cells were not randomly distributed among the sarcoplasmic components. About 98% of the total silver grains counted were present within the myocardial cells. In contrast only 5% of the total silver grains examined were observed in the interstitium (Table I-(1). These findings suggest that diltiazem, trapped by the plasma membrane, may be quickly transported into the myocardial cells and firmly bound to the cell organelles. It is noteworthy that the normalized labeling values of the plasma membrane (sarcolemma) at 3, 5 and 10min were 1.05 }0.20, 0.95 }0.19 and 0.96 }0.26, respectively. These labeling values were very low, although higher ones were expected. These low values may possibly be a result of our technique which is not sensitive enough to detect weak radioactivity of 3H-diltiazem on the plasma membrane. It is extremely important that we observed most of the silver grains in the T system, sarcoplasmic reticula and mitochondria. The normalized labeling value of the T system at 3min after injection was apparently higher that the ones at 5 and 10min after injection. Moreover, we discovered that silver grains within the sarcoplasmic reticula and mitochondria apparently increased with the passage of time (Table II). These findings suggest that diltiazem, bound to the plasma membrane, is transported through the T system and may accumulate within the sarcoplasmic reticula and mitochondria. Lullmann et al13) showed that verapamil entered myocardial cells and its intracellular concentration was 30 times higher than the extracellular concentration. Our study indicated that diltiazem also entered the myocardial cells and that the intracellular diltiazem concentration appeared to be high because of many silver grains detected within myocardial cells. Recently Vaghy et al14),15) and Matlib et al16) indicated that calcium antagonists might inhibit inorganic phosphate-induced swelling of isolated heart

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