Localization off Tritiated Digoxin in Dog Myocardium by Electron Microscopic Autoradiography

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1 Localization off Tritiated Digoxin in Dog Myocardium by Electron Microscopic Autoradiography By F. Eugene Tubbs, M.D., Lamar Crevasse, M.D., and Myron W. Wheat, Jr., M.D. With the assistance of James W. Smith Many workers have used high resolution autoradiography with success in localizing radioactive labeled substances suspected of being concentrated within the cell. 7 The majority of these studies have used tritiated thymidine in studies of protein synthesis. ' Recently tritiated norepinephrine and tritiated cholesterol have also been studied. 8 ' The present study is an attempt to demonstrate the localization of tritiated digoxin at the subcellular level in dog myocardium. In the past autoradiography has been used largely at the tissue and cellular levels. However, modifications of technique when applied to the subcellular level can give resolution of the order of. /JL. This increase in resolution is dependent upon specimen thickness, thickness of photographic emulsion, size of silver bromide crystals in the emulsion, and the energy of /? particles emitted from a given radioactive atom. With routine thin sections obtained in electron microscopic work and the utilization of low energy /? emitting isotopes as the label, intracellular localization by autoradiographic means is possible. Methods Five mongrel dogs weighing kg each were given.8 mg/kg tritiated digoxin * containing From the Departments of Medicine and Surgery, University of Florida College of Medicine, Gainesville, Florida. Supported in part by grants from the Florida Medical Research Foundation, Florida Heart Association, and Grant H-79 from the U. S. Public Health Service, Bethesda, Maryland. Received for publication August, 96. Supplied through the courtesy of Burroughs Wellcome & Company Mc/mg over an 8-hour period and were sacrificed hours following the initial dose. One-half of the calculated dose was given initially with the remainder being divided and given nine hours apart. Obviously, any fraction of digoxin which equilibrated with the cardiac tissue within the first few hours will not be evident in these studies. This schedule uniformly produced vomiting within the first hours. Dogs were given food and water ad libitum. At the end of the -hour period each dog was sacrificed using pentobarbital (Nembutal) anesthesia. Tissue was taken from the wall of the left ventricle while it was still beating and immediately fixed in buffered osmium tetroxide for one hour. The tissue was rapidly dehydrated in -second changes of alcohols of increasing strength and subsequently embedded in Epon 8 epoxy resin. After suitable curing the tissue was sectioned using a Porter- Blum ultramicrotome. All sections were cut to a thickness of approximately.6 M. Sections were mounted on parlodion-coated nichrome grids ( X AC openings) which had been coated previously with carbon. Tissue obtained from normal dogs who had not received tritiated digoxin was prepared in a similar manner and served as control. Photographic emulsion containing a monolayer of silver bromide crystals was prepared by the following method as described by Caro et al. 8 Ten grams of Ilford L- emulsion were melted in ml of distilled water in a -ml beaker at C for minutes. After complete mixing, this was cooled in an ice bath for to minutes and then left standing at room temperature for minutes. A cm diameter platinum wire loop was dipped into the mixture and was drawn out slowly. When prepared properly a thin film formed in the loop and jelled immediately. The loop was touched to the surface of grids which had been previously mounted on standard microscope slides with double-faced Scotch tape. The film fell on the grids and adhered in a firm, even fashion. Slides were then stored at C in an atmosphere of air in light-tight slide boxes containing a small amount of Drierite for time inter- Circulation Research, Volume XIV, March 96

2 LOCALIZATION OF DIGOXIN IN MYOCARDIUM 7 vals ranging from two weeks to three months. Slides were subsequently developed in Microdol-X for five minutes at C and fixed for five minutes in Edwall Quick Fix. After fixation the grids were washed in tap water for five minutes, then in distilled water for fifteen minutes and allowed to dry overnight. Following development, fixation and drying, the grids were stained for minutes in a solution of uranyl acetate containing % alcohol. This was prepared by adding cc of absolute alcohol to 7 cc of a saturated aqueous solution of uranyl acetate. After drying, grids were viewed with an RCA EMU C electron microscope at kv. Random photographs of all experimental tissues were made and compared with those of control tissue as well as grids containing no tissue which had been treated as described above. Silver grain counts were made visually before the photograph was taken and compared with counts obtained from the photograph. In order to determine how much tritiated digoxin was initially present in the tissue, and how much of the digoxin was lost during fixation and dehydration, the following studies were undertaken. Tissue from the experimental animals was cut into mg to mg wet weight specimens for analysis. These specimens were burned using the Schoniger combustion technique, 9 and the tritiated water thus formed was collected and counted in a Packard Tri-Carb liquid scintillation counter. Tissue samples at the end of dehydration were weighed, burned, and counted in the same manner. Samples of various solutions used during the dehydration procedures were also counted. The area occupied by the mitochondria and sarcomeres was estimated by cutting out these structures from ten random photographs and weighing them on an analytical balance. The remaining portions of these photographs were also weighed. The area of the mitochondria and sarcomeres is expressed as percentage of total area of photograph. These data are shown in table. Results LOSS OF LABELED DIGOXIN DURING FIXATION AND DEHYDRATION To evaluate loss of labeled digoxin during tissue processing, several parameters were measured. Table demonstrates that the number of counts lost from the tissue was similar for both methods of dehydration until higher concentrations of alcohol were reached. It is apparent that concentrations of alcohol above 8% caused a marked increase in loss Circulation Research, Volume XIV, March 96 TABLE Percentages of Total Area of Random Photograplts for Sarcomeres, Mitochondria and Other Structures Sarcomere Mitochondria Other Method for determining this area is described in the text. TABLE Effect of Long and Short Intervals of Dehydration on Leaching Out Digoxin from Labeled Tissue Alcohol concentration Abs. min Counts expressed as counts/min/ml of dehydration fluid. Wet, unfixed specimens contained counts/mg tissue per minute. TABLE Effect of Dehydration and Fixation on Number of Counts/mg Tissue per Minute Unfixed tissue 7- min dehydration sec dehydration of label in those tissues which had been dehydrated at five-minute intervals. Table clearly demonstrates the final effect of the two methods of dehydration upon the amount of labeled drug present in the tissue at the end of dehydration. In those samples subjected to five-minute intervals of alcohol dehydration, only 8% of the original amount of isotope remained as compared to % of the isotope remaining in samples dehydrated at -second intervals. LOCALIZATION OF TRITIATED DIGOXIN Our findings agree with those of Stenger and Spiro who have reviewed the ultrastructure of the cardiac muscle cell of dog and rat hearts. In summary it will be recalled that the structural unit of the myofibril is the sarcomere, defined as that portion lying between two consecutive Z lines (fig. ). The banded appearance of the sarcomere is a result of the interrelationships of thick and thin myofilaments. According to Huxley and

3 8 TUBBS, CREVASSE, WHEAT \*-z z B. / FIGURE A. Longitudinal section of relaxed sarcomere demonstrates clearly relationship of actin and myosin filaments. Note that only one-half of the I band is present on either end of sarcomere and contains only actin. The H disc contains only myosin. Small "nodules" in center of each myosin filament result in M line. The A band contains the entire myosin filament with interdigitation of actin and myosin filaments (not demonstrated here) seen in this area. (Model after Huxley and Hansen.) B. When sarcomere is contracted, band is obscured due to possible folding of actin filaments. The A band becomes quite dense, presumably due to either folding or coiling of myosin filaments attached to actin. A band = A; Z line = Z; band = J; II disc = / /; M line = M; secondary A band = A'; secondary I band = /'. Hansen, the thick filaments are thought to be myosin and the thin filaments actin. In relaxed muscle the Z lines and I bands (fig. A and B) contain only thin myofilaments, whereas the H disc and M line demonstrate only thick filaments (figs. -). The thick filaments have also been observed to have nodular thickenings at the M line (fig. A). The A band is defined as the region containing thick filaments. The interdigitation of both thick and thin filaments occurs at this region (figs. A, -). Tissue which we have observed has all been in a state of near maximal contraction. Hence the myofibrils do not demonstrate the classical banding but assume the appearance described by Gilev of secondary A and H bands with faint or absent secondary I bands (figs. IB, -). Small cross bridges between the thick and thin filaments are often seen in the region of the A band (fig. ). As a rule, rather than an exception, the relationship of one mitochondrion per sarcomere was found (figs. -). Large numbers of unattached, electron-dense cytoplasmic granules, thought by Fawcett and Selby to be glycogen were seen in all sections (fig. ). When the emulsion is applied correctly, it consists of a monolayer of silver bromide crystals. ^ thick. After the photographic process, we are looking no longer at the silver bromide crystal, but at a filament of silver which starts from the latent image and grows out of the crystal (fig. A and B). The silver filaments will assume two basic forms. The first will be a comma form (fig. ) and the second a slightly coiled filament form (figs.,, 6). The measured length of these silver filaments ranges from. /x to. p. with an average length of about. //.. Using the Circulation Research, Volume XIV, March 96

4 LOCALIZATION OF DIGOXIN IN MYOCARDIUM data of Caro we have determined the point source of radioactivity to be at the head of the comma form, at the middle of the filament form, and at the estimated center of other more complex solid and coiled forms. Figures,, and demonstrate the various types of silver grains observed and their rela- 9 tionships to the ultrastructure of the dog myocardium. These sites of activity were best appreciated after two months of exposure. Sites of isotope activity were localized at the A band, Z line, H disc, I band, and mitochondria (figs. -). Table shows silver grain counts in a FIGURE Two nuclear tracks are present. One overlies secondary A band, the second overlies H disc. Close relationship of mitochondria to sarcomeres is apparent. Note the comma form of silver crystals. Secondary A band = A; H disc = H; Z line = Z; mitochondria = A/; ghjcogen granules = C; nuclear tracks = N. Original magnification X,. Circulation Research, Volume XIV, March

5 TUBBS, CREVASSE, WHEAT > *< FIGURE Several nuclear tracks overlie A bands of these myofibrils. One track is in close association with Z line while another clearly overlies a mitochondrion. Silver crystals assume a more filamentlike appearance. Z line = Z; secondary A band = A; mitochondria = M; nuclear track N. Original magnification X,. i X i>. area of random sections. In such sections of labeled digoxin treated tissue, the average number of grains found was 8. Counts were also made in a similar manner using nonlabeled tissue and blank grids. Both of these showed average counts well below that of the tissue containing labeled digoxin. Table demonstrates the distribution of silver grains in the cardiac muscle cell. Five grids were counted visually in the electron microscope. After the data had been obtained, random photographs were made, and the percentage of counts localized over a given area in photographs was compared with results Circulation Research, Volume XIV, March 96

6 LOCALIZATION OF DIGOXIN IN MYOCARDIUM obtained directly. The large difference in total number of counts per grid is a reflection of the amount of tissue visible on any individual grid. Control grids were also counted in this manner. Interpretation of Results Few data are available concerning cellular localization of cardiac glycosides. Harvey and Pieper studied intracellular distribution of C- digitoxin in isolated, perfused guinea pig heart and found that after differential centrifugation of the heart homogenate, the greatest amount of radioactive label was found in the aqueous phase. However, when the vascular FIGURE Silver crystals overlying labeled structures assume a more complex nature with comma, filament and solid forms shown here. Site of nuclear activity is below center of solid silver crystal at labeled secondary A band. Secondary A band = A; Z line = Z; mitochondria = M; nuclear tracks = N. Original magnification X,. Circulation Research, Volume XIV, March 96

7 TUBBS, CREVASSE, WHEAT TABLE Silver Grains Present in Randomly Selected Sites, Square Micra in Area r CRYSTAL 'EMULSION ) TISSUE SPECIMAN SUPPORTING PLASTIC 6;* H-OIGOXIN -SILVER GRAIN FIGURE A. Latent image which is formed by collision of a p particle with a crystal of silver bromide occurs during exposure time. With thin tissue sections and a monolayer of silver bromide overlying tissue, the chance of a point source of radioactivity resulting in latent image production is almost %. B. After development silver filament which has grown from latent image remains over site of radioactivity (redrawn after Cam). space of guinea pig hearts was washed free of label, they found that the centrifuged debris fraction now contained the greatest amount of label. The mitochondrial fraction of this debris contained only one-fourth the concentration of labeled glycoside. If one assumes that the debris fraction is largely composed of disrupted sarcomeres, then the present study is in good agreement as to the localization of the labeled drug. Furthermore, during the preparation of this manuscript, Smith FIGURE 6 An electron micrograph of a portion of a grid incubated with isotope and photographic emulsion but no tissue. The variously formed nuclear tracks are shown. Original magnification X,7. H-digoxin Control tissue Blank grids 8 (-6) (-6) (-7) Upper values represent average number counted in observations. Lower values represent maximum and minimal values. Exposure time, two months. and Fozzard ie have reported findings similar to ours localizing this tritiated compound to the A band of the sarcomere. However, it should be emphasized that the technique of autoradiography allows identification of the isotope only. The amount of label which is represented by the intact drug or by its breakdown products cannot be determined with this technique. Our data demonstrate that the labeled glycoside is found in the myocardial cell, largely in association with the myofibril. It is of interest that a high percentage of label was localized at the A band of the sarcomere. It should be pointed out, however, that the sarcomeres are all in a contracted state with the A band constituting a large percentage of the sarcomere. This localization of label over the sarcomere is considered more meaningful in light of the estimated per cent total area of the sarcomere with respect to other structures. Whereas % of the tissue area was occupied by sarcomere, approximately 7% of all nuclear tracks were counted over the sarcomere. If the distribution of these tracks were on a random basis only, one would expect a smaller number of counts to be over the sarcomere and a corresponding increase in counts over remaining structures. Evaluation of the effects of fixation and dehydration on the location of the labeled digoxin is extremely difficult. Data presented demonstrate clearly that with prolonged alcohol dehydration, a significant amount of label is leached out of the tissue. Acetone has been used for dehydration in an attempt to lessen this problem but is highly destructive to the tissue. As long as tissue specimens are quite small, no change in histologic appearance of the tissue was noted with this rapid dehycirculation Research, Volume XIV, March 96

8 LOCALIZATION OF DIGOXIN IN MYOCARDIUM TABLE Location of Silver Grains and Their Distribution in the Cardiac Muscle Cell Grid Total Counts Total sarcomere A band H disc Z band Mitochondria Endoplasmic reticulum Connective tissue Vascular spaces Intercalated disc Sarcolemma Percentage of total counts Sarcomere Mitochondria Other Percentage of sarcomere counts A band H disc Z band l Control Blank. The differences seen in total number of counts/grid are a reflection of the amount of tissue able to be seen on any individual grid. All percentages are rounded off to the nearest whole number. Exposure time, two months. dration method. With the rapid dehydration procedure, the excessive loss of label was prevented to a great extent but was not completely circumvented. Possibly this objection may be overcome by the use of embedding media which do not necessitate such complete dehydration. The large loss of label associated with fixation is predominantly a washing-out phenomenon and is substantiated by the data previously presented of Harvey and Pieper. ln If we compare the activity of wet, unfixed tissue to that of fixed, dehydrated tissue, it is immediately apparent that a large amount of activity has been lost during tissue preparation. The application of autoradiographic techniques to electron microscopy offers a relatively simple means of localizing various compounds at the subcellular level. If the compound has a definite location within the cell such as the nucleus or cell membrane, evaluation of this type of data is straightforward. However, in this study we have attempted to localize a compound which has no known specific site of high concentration within the myocardial cell. Our data demonstrate that the Circulation Research, Volume XIV, March 96 digoxin molecule is indeed found predominantly associated with the myofibril and associated mainly with the A band area. Summary Five mongrel dogs were given.8 mg/kg of tritiated digoxin over a -hour period. Left ventricular myocardium was excised and subsequently fixed, dehydrated, and embedded in Epon 8 epoxy resin. Tissue was sectioned and coated with a monolayer of photographic nuclear emulsion and exposed for varying time intervals up to three months. After development these autoradiographs were viewed with the electron microscope. It was found that of the total number of nuclear tracks counted, 7% were found to overlie the sarcomere of the myofibril, 7% to overlie the mitochondria, and 8% to overlie other structures. Of those tracks associated with the sarcomere, 7% were found to overlie the A band. Evaluation of the data obtained and the techniques used are presented. Acknowledgment We wish to thank Dr. William P. Callahan for his

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