Normal and hypertrophic growth of the rat heart: changes in cell dimensions and number B. KORECKY Department
K. RAKUSAN Faculty
of Medicine,
Normal and hypertrophic charges in cell dimensions and rzunzber. Am, J. Physiol. 234(2): H123-H128, 1978 or Am. J* Physiol.: Heart Circ. Physiol. 3(2): H123-H128, 1978. -The length and width of enzymatically isolated individual rat cardiac myocytes were concurrently measured during normal and stimulated cardiac growth. In normal rats weighing between 75 and 750 g the length and width increased by 64 and 68% while their ratio remained constant ka. 5.3). The cell volume, calculated on the basis of a cylindrical model, increased almost 5 times. The rates of increase in the volume of an average myocyte and in left ventricular mass were found to be similar, indicating that normal myocardial growth could be explained by hypertrophy of existing myocytes and no proliferation would be required. In cardiomegaly induced by aortic constriction in the adult rat, an increase in cell volume was observed while no significant changes in the length-to-width ratios could be detected. The cell volumes of the hypertrophic hearts corresponded to those observed in hearts of similar weight obtained from larger normal rats and the stimulated cardiac growth could also be explained solely by hypertrophy of existing cells. KORECKY,
growth
B.,
of’ the
AND
ml
K.
AND
of Physiology,
RAKUSAN.
heart:
enzymatic isolation of adult cardiac cells; length, width, and volume of cardiac myocytes; allometric relationships between cell dimensions and body weight
ARE SURPRISINGLY FEW quantitative data in the literature dealing with dimensional changes of cardiac muscle cells during the normal postnatal growth of the mammalian heart (1, 2, 10, 12). In addition, all the above data deal with the relationship between the increasing cellular width, i.e., muscle fiber diameter, and the growth of the heart which, as an organ, is closely related to the growth of the whole body. The length of the cardiac myocytes was measured in adult dog hearts (8>, but no data are available for the developing myocardium and no concurrent measurement of cellular width and length has been reported. Recently successful attempts have been made to disaggregate cardiac tissue of adult rats by enzymatic and mechanical disruption of the interstitium in order to obtain pure samples of individual cardiac myocytes with minimally damaged sarcolemma (3). These individual myocytes obtained from adult hearts showed reasonable morphological quality and exhibited longterm beating, If representative samples of myocyte populations of acceptable morphological quality could be obtained during the life-span of the rat, then the relationship beTHERE
University
of Ottawa,
Ottawa
Canada,
KlN
9A9
tween the length and width of the growing myocytes could be measured and their volumes could be estimated. Furthermore, by correlating the rate of growth of an average myocyte with that of the heart or of the body, potential changes in the total number of myocytes during the normal growth of the rat could be detected. In a similar way, the cellular dynamics of stimulated cardiac growth could be also investigated. MATERIALS
AND
METHODS
Normal male Sprague-Dawley rats (Bio-Breeding Laboratories) weighing between 75 and 750 g were anesthetized with Nembutal (30 mg/kg) and heparinized (10 mg/kg). The abdominal cavity was exposed and the animal was bled from the abdominal aorta. The thorax was then opened and the aortic arch was cannulated with a X-gauge needle, which was filled with saline and closed with a rubber stopper. Its tip was then advanced to a point 2-3 mm above the aortic valve and secured there by means of a surgical thread (no. 000) around the ascending aorta. The whole heart with the cannula was then removed, submerged in saline, the stopper subsequently removed, and the remaining blood washed from the heart by gentle massage. The stopper was then replaced and the cannulated heart mounted on a perfusing system. This consisted of a double-jacket muscle bath, the outer portion of which was connected to a thermostatic bath (Haake) through plastic tubings. The muscle bath was filled with 60 ml of calcium-free Ringer (143.1 mM NaCl, 3.6 mM KCl; 1.2 mM MgSOd, 5.5 mM glucose, and 3 mM HEPES, at pH 7.4 adjusted by 0.5 N NaOH). Pure oxygen was bubbled through a sintered glass filter at the bottom of the bath. The bath outflow was connected via Teflon tubing through a peristaltic pump (Harvard n o. 1203) to a plexiglass block with a predrilled passage (l/8 inch), containing the tip of a needle telethermometer (Yellow Springs) sealed inside the passage through which the perfusate passed. A threeway stopcock was attached to the outflow part of the block, to which a mercury manometer was connected and on which the cannula with the heart could also be mounted. The whole perfusion system was first primed with Ca-free Ringer solution, then the heart with the cannula was connected, and retrograde perfusion of the heart was started at a pressure of 40-60 mmHg. The left ventricle was then opened by a small stab wound to
H123
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H124 allow drainage, should the aortic valve become incompetent. The fnst 20 ml of perfusate containing residual blood were discarded. The heart was positioned above the muscle bath and the perfusate was recirculated At this point, 20 ml of the above into the system. solution containing 0.3% hyaluronidase (Sigma; type I) and 0.15% collagenase (Sigma; type I) buffered to pH 7.4 were introduced into the muscle bath. Subsequently the hearts were retrogradely perfused through the coronary vessels at 36°C with 60 ml of the latter medium in a closed recirculating system for approximately 30-N min at a flow rate which was initially adjusted to about 5-7 ml/min. This was changed to 7-10 ml/min. after the first 5 min of perfusion. The perfusion pressure was kept below 100 mmHg. Pure OeI was bubbled through the perfusion medium. Two criteria were used to make sure that the heart was being digested: the softening of the heart was occasionally checked by touch and the perfusion pressure was monitored. When this pressure fell below 10 mmHg the perfusion was stopped and the heart was removed from All subsequent manipulations the perfusion system. were done at room temperature. The softened heart was placed on a Petri dish, the left ventricle was cut off and the right ventricle with the septum was discarded. The left ventricle was transferred into a Petri dish containing Ca-free Ringer solution and minced with fine scissors into small pieces. Then it was transferred into a siliconized flask and gently shaken until the upper layer appeared cloudy (5-10 min). After the larger pieces of tissue had settled down, the supernatant was poured into siliconized test tubes and checked under a phase-contrast light microscope for its quality and yield. Two to three fractions were subsequently obtained, in a similar way, from the remaining suspension. Aliquots from gently stirred individual fractions were transferred with a Pasteur pipet into a Petroff-Hauser bacteria counting chamber and observed under phase contrast in a Zeiss RA microscope utilizing ~630 or ~1,000 magnification. The image was displayed on a viewing screen, and the widths and the lengths of individually isolated cardiac myocytes were measured with a ruler. Only cells that appeared intact, did not branch, and showed a distinct cross-striation were considered. The largest diameter of the cells was measured, and their length was estimated after averaging the irregularities of the intercalated discs at the discretion of the observer. In each cell the length and width were measured, their ratio calculated, and the volume determined on the assumption of a cylindrical configuration. The spontaneous contractions were of low frequency and the beating cells remained slack for about 10 s, which is enough for measurement. In each heart 100 cells were measured since, in a preliminary study, no significant differences were found among results obtained from 100, ZOO, 300, and 400 cells from the same heart. The average sarcomere lengths were estimated by direct measurement of a series of 20 sarcomeres on a clearly identifiable myofibril in one cell, and the mean value was then calculated. A total of 15-20 cells in each heart was investigated.
B. KORECKY
AND
K.
RAKUSAN
Cardiac hypertrophy was produced in normal male Sprague-Dawley rats (Bio-Breeding Laboratories), with body weights between 340 and 360 g, by constricting the abdominal aorta below the diaphragm with a silver ring. This technique has been described in detail elsewhere (7). A group of animals with a similar initial body weight was kept as controls. Both groups were sacrificed 2-3 wk after the operation, and the cardiac myocytes were isolated as above. To compare the ventricular weights (heart weights) of the two groups, each isolated and cannulated heart was trimmed of adhering tissues and submerged in Ringer solution. Then the stopper was removed, the remaining blood washed out by gentle massage, the liquid squeezed out of the heart, the stopper replaced, and the whole system weighted. After retrograde perfusion was completed the heart was transsected between the atria and ventricles, the cannula filled with liquid, and the original stopper reintroduced. The weight of the cannula with the atria was then subtracted from the original weight of the whole system, and the difference was taken as an estimate of the weight of the heart, excluding the atria, in normal animals and animals with aortic constriction. The morphometric analysis of the isolated cells was the same as above. RESULTS
The changes in dimensions of average left ventricular myocytes during the postnatal period between 1 and 8 mo of age in normal rats are summarized in Table 1. The average body weight increased approximately eightfold, from 81 g to 650 g Both cellular length and width increased by about 609& their ratio remaining remarkably constant through this period. The average cell volume increased from 14 x lO:j to 67 x 10” pm”, representing an almost fivefold increase. The morphometric data obtained from hearts of individual animals are displayed as a function of body weight in Fig. 1. If plotted on a double-logarithmic scale, the increases in width, length, and volume follow a straight line with respect to increasing body weight of the animals. Consequently, these relationships may be expressed by individual allometric equations, which wiI1 have a general form of y = 6x”, where y is the respective cell dimension, x is the body weight, b is a constant, and a is the coefficient expressing the slope 1. Body weights and cell dimensiorzs in five age groups of normal rats - -_---
TABLE
No. of FZats ~~--.
B&y
Wt, &
Cell
Length, tim ---- -.-.-. - --
Cell
Width, Pm
Cell Volume, pm” X 10:’
Length/Width --~
6
81.2k1.7
78.1H.3
14.9kO.5
5.3kO.11
14.421.2
9
159.8k7.4
84.8k2.1
16.7kO.4
5.19*0.06
19.7k1.4
9
365.9258
103.Od.O
20.550.2
5.14kO.06
35.621.0
463.5~12.1
119.922.2
22.2e0.3
5.48kO.10
48.4k2.1
647.8d3.0
127.5d.7
25.1kO.5
5.29&0.11
67.2k3.2
11 9 Values
are averages
t SEM.
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GROWTH
OF
28 24
CARDIAC
H125
MYOCYTE TABLE 2. Relationship between. cell dimensions and body weight calculated according to allometric equation ------------b tk r
- CELL WIDTH
20
____I”---^__--
0.253 Cell length, 24.49 Pm 4.78 0.251 Cell width, Pm 0.746 Cell volume, 0.47 pm;’ X 10:’ p-----__c____-Relationship between cell dimensions (y) grams, calculated according to the allometric constant b is the value of y when x is equal slope of the line, and its value indicates the to r; r = correlation coefficient; SEE = estimate.
80 70
- CELL VOLUME
0.942
0.0635
0.948
0.0596
0.961
0.1541
and body weight (x1 in equation: y = bx”. The to 1, constant cy is the growth ofy in relation standard error of the
TABLE 3. Body weights, heart weights, and cell dimensions in normal rats and in rats with aortic stenosis -~~_ -..-- -~. ----~-~. _.~
w3
x103
SEE
.----
20 15 10
B+
Wt,
-
a
~-. 75
100
150 200 300 400 BODY WEIGHT in g
600
FIG. 1, Mean values 295% confidence limits ( dashed lines) describing relationship between body weight and average dimensions of left ventricular myocytes in normal rats.
of’ the linear relationship and thus also the relative rate of growth with respect to body weight. The numerical values of the constants for the allometric equations are given in Table 2, The rate coefficients (a) for cell length and cell width are almost identical, which is additional evidence indicating a harmonic length-towidth relationship of normal growth of the cardiac myocytes. The high correlation coefficients and relatively small standard errors indicate a close relationship between the growth of the body and the growth of the basic morphological unit of the heart-the myocyte. In the case of stimulated cardiac growth observed after experimental aortic stenosis (Table 3), the average length, width, and volume of the left ventricular myocytes were significantly increased when compared to those of the normal control rats of similar body weight. As in the case of normal growth, there was no significant change in the length-to-width relationship. The above increments in cellular dimensions in hypertrophied hearts correspond to those that could be expected when the heart becomes larger due to normal growth, as illustrated in Fig. 2; When these average cell volumes are displayed on the growth chart of the normal cell volume-to-body weight relationship, we can see that cell volumes are higher in rats with aortic stenosis (hypertrophy) than in control rats of similar body weight. If we, however, compare the hypertrophied hearts with those of normal but heavier rats with the same heart weight (normal), the average volume of the myocytes appears to be the same. The relative frequency distribution of cell length, width, and volume of normal and hypertrophied hearts is disslaved in Fig. 3. These data are based on measure-
-..
Heart mR
Wt mg1100 IZBW
L-gth, *rn
---~-
Width, Pm
Len hl Wi $ th
Volume, pm” x
10"
--__
Controls (n = 11)
463.5 212.1
1143 k51
246 *5
119.9 k2.2
22.2 20.3
5.48 kO.10
48.4 k2.1
Stenosis (I?. = 11)
473.2 k 10.3
1381 229
293 +6
128.8 k2.4
25.1 LO.4
5.22 20.07
66.6 k3.2
SignifiNS coo1 coo1 1.02 coo1 NS -coo1 cance,--- ----P _ .~--~_ Values are averages ,+ SEM. BW = body weight, NS = not significant.
80
FIG. 2. Mean values +SEM of average volume of left ventricular myocytes in a group of control rats and rats with expFrimenta1 aortic stenosis (hypertrophy) plotted on growth chart expressing normal relationship between body weight and average volume of myocyte (see Fig. 1). Values for hypertrophic hearts are then extrapolated (dashed lines) to body weight, which would correspond to that of older normal rats having similar heart weight (normal).
ments of 100 cells in each heart and the values for individual classes were subsequently averaged for the whole group. It appears that in the case of hypertrophy, there is a general trend of increasing the size in all chosen myocytes, as represented by the arbitrarily classes.
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H126
B. KORECKY
RELATIVE
FREQUENCY
DISTRIBUTION
-COrJTRROL ---HYPERTROPHY
%
LENGTH
%
FIG. 3. Mean values of arbitrarily selected classes of cardiac myocytev with respect to their length, width, and volume in a group of control rats and rats after aortic constriction (hypertrophy). Volumes are expressed in units of pm x 10%
DISCUSSION
Enzymatic disruption of the left ventricular myocardium in the rat provides a yield of individual cells that shows morphological characteristics very similar to those observed in cells from” histological sections of the normal left ventricular myocardiumr The fact that these cells are isolated offers the advantage of inspection of the whole cell under the microscope. Approximately 50% of the observed cells exhibited normal morphology, while the remainder showed varying degrees of damage and were therefore not consider&d. since the final yield amounted to approxi mately half of the mass of the original tissue, we may assume that our sample of isolated cells contained ?O-30% of the original population of myocytes This relatively low yield could be considered adequate for further measurement, provided that the final sample may be shown to be representative of the original cell population of the left ventricle. We have no direct experimental proof that our sample is indeed representative, but some circumstantial evidence suggests that it may be so. First, no significant differences were found between momhometric measurements obtained from different fractions during the isolation procedure. Second, when samples were taken from different parts of the left ventricle after perfusion, no significant difference in morphometry of myocytes was observed. Third, when individual parts of the left ventricle were taken from one rat and the cells isolated by incubation in the enzyme solution with concurrent application of gentle shear stress (shaking or rotation), the morphometric data obtained after these isolation procedures did not significantly differ from those obtained after isolation by the retrograde perfusion technique. The selection of normal cells for measurement under the microscope depends on the decision of the observer, and the results may therefore be affected by an individual bias. All the presently reported data were those measured by one of us (B.K. ); measurements obtained in the same samples by other investigators in our
AND
K. RAKUSAN
laboratory did not differ significantly from those reported in this study. The average length of the sarcomeres after isolation, as determined concurrently in four to six hearts of each group, under the light microscope, was found to be 1.8 pm, which is approximately 0.1 pm less than was estimated for rat papillary muscle at slack length (4, 6) in different preparations. This degree of contracture is unfortunately unavoidable and occurs most probably during the enzymatic disruption of the heart with collagenase and hyaluronidase, which require CaZ+ for maintaining this activity, since, after addition of Ca buffer to the perfusing solution, the hearts could not be digested even after several hours of perfusion. Addition of EGTA (0.25 PM- 1 mM) to the final suspension of isolated cells did not abolish the existing partial contracture. Since the average sarcomere lengths, as measured under the light microscope, did not differ between myocytes of individual age groups, we may assume that this technique causes a constant degree of contracture which appears to be independent of the age and weight of the animal. The width of isolated adult cardiac myocytes (20-25 pm) appears to be greater than that reported by other investigators who utilized standard histological techniques (12-16 pm) (1, 2, 10, 12). This difference may be explained by a combination of the following factors: the isolated myocytes are in partial contracture and they are devoid of interstitium which may have restricted them. They probably exhibit a certain degree of swelling, since their sarcolemmas would be at least temporarily damaged during the separation from neighboring cells (e.g., cleavage of nexuses). All these factors would probably lead to increase of cell diameter. Since the cells have settled down in the chamber, it is likely that the greatest diameter will be the one which will yield itself for measurement. On the other hand, the preparation of myocardium for histology usually involves dehydration and subsequent shrinkage, which may lead to a decrease of cell diameter. The lengths of isolated adult cardiac cells are in the range of 100428 pm. We are not aware of any histological measurement of cell length in the rat but Laks et al. (8) reported an average value for adult dog cardiac myocytes of 71 pm. It seems therefore that both the width and the length of isolated myocytes are larger than those reported in histological preparations. It is likely that neither of the two techniques yields values that truly reflect the cellular size in vivo. Results based on histological techniques may be distorted by shrinkage, poor sampling, and deviation from cylindrical configuration of muscle cells. On the other hand, isolated myocytes may increase in size, due to absence of surrounding connective tissue and swelling. In addition, a certain selection could not be completely excluded because of the relatively low final yield. The only histological data dealing with postnatal increase of the width of cardiac myocytes in the rat (12) showed an increase from 6 pm at birth to 10 pm at 30 days and to 16 pm in adult animals. In the present study a similar relative increase (65%) in width was observed between the youngest group (ca. 30 days
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GROWTH
OF
CARDIAC
H127
MYOCYTE
old) and the oldest group, which was ea. 8 mo of age. The constancy of the relationship between length and width of cardiac myocytes during the above period indicates the harmonic growth of the cell, which, to our knowledge, has not previously been reported. Our data also indicate that the dimensions of myocytes increased in proportion to the increasing body weight over a range of 75-750 g. It would be tempting to calculate the total number of myocytes constituting the growing left ventricle by dividing its mass by the volume of its average myocyte and determine the total number of myocytes. Unfortunately, because of problems inherent in the technique of cell isolation, the left ventricular weight and the cell size could not be determined in the same heart. However the rate of growth of the mass of the left ventricle and the rate of growth of its myocytes may be compared indirectly taking into consideration the following. First, the average cell volume (V) may be related to the total body weight (BW) as V = 0,475 BW*=7”6 (see Table 2). Second, the left ventricular weight (LVW) may be also calculated as a product of average myocyte volume (V), total number of myocytes (NJ, and specific weight of cardiac tissue (D) divided by the fraction of the left ventricle formed by myocytes only (F); therefore, LVW = V x N x D/F. Finally, the directly determined left ventricular weight was found to be related to the total body weight according to the following allometric equation: LVW (mg) = 5.662 BW0v7-15, as determined previously in a large number of normal animals from our colony between 40 and 800 g in body weight. By comparing the values for the exponent Q! in the above two equations (0.746 and 0.745), we can conclude that the rates of growth of cell volume of the mass of the left ventricle were identical. If we assume a constant ratio between myocytes and interstitium and constant density (D/F = constant), the total number of myocytes constituting the left ventricle must have remained unchanged during the above age period. In case of stimulated cardiac growth, which was induced by increased afterload due to experimental aortic constriction, cellular hypertrophy was also observed by direct morphometric measurements from isolated myocytes. The larger cellular volume observed in this type of hypertrophy was similar to the volume of myocytes of normal rats of1 arger body weight but with heart weigh ts similar to the anim .als wi th experim ental cardiomegaly (see Fig. 2). Consequently, a simple cellular hypertrophy with no concomitant increase in the total number of myocytes would be the most probable explanation for the stimulated cardiac growth of adult rats as well.
However, it should be pointed out that the cardiac hypertrophy in our experimental group was of relatively mild degree: the final heart weight in animals with aortic constriction did not exceed the heart weight in normal animals with the highest body weight. Some of our results, especially those concerning the symmetrical cell growth may not necessarily apply to the cases of excessive cardiac hypertrophy and/or cardiac dilatation. Nevertheless, the conclusions that we derived from our results on normal and stimulated cardiac growth agree with those reported by others (5, 9, 13) who studied the normal and stimulated cardiac growth in humans by determining the density of cardiac muscle muclei in histological preparations from normal and hypertrophic hearts. From our present results we may conclude that normal cardiac growth in the rat fro& weaning to adulthood is almost entirely due to the growth of existing cells, i.e., cellular hypertrophy. In addition, the stimulated growth of the adult heart was also accomplished by accelerated growth of existing myocytes. There is, however, some experimental evidence that the proliferation of cardiac myocytes as a mode of heart growth is present during the first 3-4 wk of postnatal life in the rat (12, 13). During this period, the rate of growth of the heart is faster than later on, and in the case of the left ventricle, it may be related to the increase in body weight as LVW = 2.586 BW1.0g2 (11). Unfortunately, we have no morphometric data on cardisc myocytes for this age because of technical difficulties associated with their isolation. However, if we extrapolate the cell volume-to-body weight relationship established for older animals into this early postnatal period, then the rate of heart growth (a! = 1.092) becomes faster than the rate of cell volume growth (a = 0.745). Assuming an average body weight at birth of 5 g and at 3 wk of 40 g, a twofold increase in the total number of myocytes would be predicted to explain the normal increase in cardiac mass for the above period. The extrapolation of the cell volume-to-body weight relationship into this early postnatal period is speculative, but it is interesting to note that a similar increase of the total number of myocytes was postulated for the first 3 wk of postnatal life in the rat by Hort (5) in his morphometric studies based on evaluation of nuclear densities with respect to heart weight. This Medical
Received
study was supported by Ontario Research Council of Canada.
for publication
27 June
Heart
Foundation
and
1977.
REFERENCES
ARAI,
S., A. MACHIDA, AND T. NAKAMURA. Myocardial structure and vascularization of hypertrophied hearts. T&&u J. Exptl. Med. 95: 35-54, 1968. 2. ASHLEY, L. M. A determination of the diameters of ventricular myocardial fibers in man and other mammals. Am. J. Anat. 77: 325-364, 1945. 3. BERRY, M. N., D. S. FRIEND, AND J. SCHEUER. Morphology and metabolism in intact muscle cell isolated from adult rat heart. Circdation Res. 26: 679-687, 1970. 1.
4. GRIMM, A. F., K. V. KATELE, S. A. KLEIN, AND H. L. LIN. Growth of the rat heart. Left ventricular morphology and sarcomere lengths. Growth 37: 189-208, 1973. 5. HORT, W, Quantitative histologische Untersuchungen an wachsenden Herzen. Arch. Pathol. Anat. Physid. 323: 223-247, 1953. 6. JEWELL, B. R. A reexamination of the influence of muscle length on myocardial performance. Circutation Res. 40: 221230, 1977. 7. KORECKY, B., K. RAKUSAN, AND 0. POUPA. Experimental car-
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HE8 diomegaly. Methods Achiev. Exptl. PathoZ. 2: 158-171, 1967. 8. LAKS, M. M., M. J. NISENSON, AND H. J. SWAN. Myocardial cell and sarcomere lengths in the normal dog heart. Circulation Res. 21: 671-678, 1967. 9. LINZBACH, A. J. Mikrometrische und histologische Analyse hypertropher menschlicher Herzen. Arch. PathoZ. Amt. PhysioE. 314: 534-594, 19.17. 10. MUNELL, J. F., AND R. GETTY. Nuclear lobulation and amitotic division associated with increasing cell size in the aging canine myocardium. J. Gerontol. 23: 363-369, 1968.
B. KORECKY
AND
K.
RAKUSAN
11. RAKUSAN, K., B. KORECKY, 2. ROTH, AND 0. POUPA. Development of the ventricular weight of the rat heart with special reference to the early phases of postnatal ontogenesis. Physiol. Bohemoslov. 12: 518-524, 1963. 12. RAKUSAN, K., AND 0. POUPA. Changes in the diffusion distance in the rat heart muscle during development. Physiol. Bohemoslov. 12: 220-227, 1963. 13. ZAK, R, Cell proliferation during cardiac growth. Am. J. Cardial. 31: 211-219, 1973.
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K. RAKUSAN Faculty
of Medicine,
Normal and hypertrophic charges in cell dimensions and rzunzber. Am, J. Physiol. 234(2): H123-H128, 1978 or Am. J* Physiol.: Heart Circ. Physiol. 3(2): H123-H128, 1978. -The length and width of enzymatically isolated individual rat cardiac myocytes were concurrently measured during normal and stimulated cardiac growth. In normal rats weighing between 75 and 750 g the length and width increased by 64 and 68% while their ratio remained constant ka. 5.3). The cell volume, calculated on the basis of a cylindrical model, increased almost 5 times. The rates of increase in the volume of an average myocyte and in left ventricular mass were found to be similar, indicating that normal myocardial growth could be explained by hypertrophy of existing myocytes and no proliferation would be required. In cardiomegaly induced by aortic constriction in the adult rat, an increase in cell volume was observed while no significant changes in the length-to-width ratios could be detected. The cell volumes of the hypertrophic hearts corresponded to those observed in hearts of similar weight obtained from larger normal rats and the stimulated cardiac growth could also be explained solely by hypertrophy of existing cells. KORECKY,
growth
B.,
of’ the
AND
ml
K.
AND
of Physiology,
RAKUSAN.
heart:
enzymatic isolation of adult cardiac cells; length, width, and volume of cardiac myocytes; allometric relationships between cell dimensions and body weight
ARE SURPRISINGLY FEW quantitative data in the literature dealing with dimensional changes of cardiac muscle cells during the normal postnatal growth of the mammalian heart (1, 2, 10, 12). In addition, all the above data deal with the relationship between the increasing cellular width, i.e., muscle fiber diameter, and the growth of the heart which, as an organ, is closely related to the growth of the whole body. The length of the cardiac myocytes was measured in adult dog hearts (8>, but no data are available for the developing myocardium and no concurrent measurement of cellular width and length has been reported. Recently successful attempts have been made to disaggregate cardiac tissue of adult rats by enzymatic and mechanical disruption of the interstitium in order to obtain pure samples of individual cardiac myocytes with minimally damaged sarcolemma (3). These individual myocytes obtained from adult hearts showed reasonable morphological quality and exhibited longterm beating, If representative samples of myocyte populations of acceptable morphological quality could be obtained during the life-span of the rat, then the relationship beTHERE
University
of Ottawa,
Ottawa
Canada,
KlN
9A9
tween the length and width of the growing myocytes could be measured and their volumes could be estimated. Furthermore, by correlating the rate of growth of an average myocyte with that of the heart or of the body, potential changes in the total number of myocytes during the normal growth of the rat could be detected. In a similar way, the cellular dynamics of stimulated cardiac growth could be also investigated. MATERIALS
AND
METHODS
Normal male Sprague-Dawley rats (Bio-Breeding Laboratories) weighing between 75 and 750 g were anesthetized with Nembutal (30 mg/kg) and heparinized (10 mg/kg). The abdominal cavity was exposed and the animal was bled from the abdominal aorta. The thorax was then opened and the aortic arch was cannulated with a X-gauge needle, which was filled with saline and closed with a rubber stopper. Its tip was then advanced to a point 2-3 mm above the aortic valve and secured there by means of a surgical thread (no. 000) around the ascending aorta. The whole heart with the cannula was then removed, submerged in saline, the stopper subsequently removed, and the remaining blood washed from the heart by gentle massage. The stopper was then replaced and the cannulated heart mounted on a perfusing system. This consisted of a double-jacket muscle bath, the outer portion of which was connected to a thermostatic bath (Haake) through plastic tubings. The muscle bath was filled with 60 ml of calcium-free Ringer (143.1 mM NaCl, 3.6 mM KCl; 1.2 mM MgSOd, 5.5 mM glucose, and 3 mM HEPES, at pH 7.4 adjusted by 0.5 N NaOH). Pure oxygen was bubbled through a sintered glass filter at the bottom of the bath. The bath outflow was connected via Teflon tubing through a peristaltic pump (Harvard n o. 1203) to a plexiglass block with a predrilled passage (l/8 inch), containing the tip of a needle telethermometer (Yellow Springs) sealed inside the passage through which the perfusate passed. A threeway stopcock was attached to the outflow part of the block, to which a mercury manometer was connected and on which the cannula with the heart could also be mounted. The whole perfusion system was first primed with Ca-free Ringer solution, then the heart with the cannula was connected, and retrograde perfusion of the heart was started at a pressure of 40-60 mmHg. The left ventricle was then opened by a small stab wound to
H123
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H124 allow drainage, should the aortic valve become incompetent. The fnst 20 ml of perfusate containing residual blood were discarded. The heart was positioned above the muscle bath and the perfusate was recirculated At this point, 20 ml of the above into the system. solution containing 0.3% hyaluronidase (Sigma; type I) and 0.15% collagenase (Sigma; type I) buffered to pH 7.4 were introduced into the muscle bath. Subsequently the hearts were retrogradely perfused through the coronary vessels at 36°C with 60 ml of the latter medium in a closed recirculating system for approximately 30-N min at a flow rate which was initially adjusted to about 5-7 ml/min. This was changed to 7-10 ml/min. after the first 5 min of perfusion. The perfusion pressure was kept below 100 mmHg. Pure OeI was bubbled through the perfusion medium. Two criteria were used to make sure that the heart was being digested: the softening of the heart was occasionally checked by touch and the perfusion pressure was monitored. When this pressure fell below 10 mmHg the perfusion was stopped and the heart was removed from All subsequent manipulations the perfusion system. were done at room temperature. The softened heart was placed on a Petri dish, the left ventricle was cut off and the right ventricle with the septum was discarded. The left ventricle was transferred into a Petri dish containing Ca-free Ringer solution and minced with fine scissors into small pieces. Then it was transferred into a siliconized flask and gently shaken until the upper layer appeared cloudy (5-10 min). After the larger pieces of tissue had settled down, the supernatant was poured into siliconized test tubes and checked under a phase-contrast light microscope for its quality and yield. Two to three fractions were subsequently obtained, in a similar way, from the remaining suspension. Aliquots from gently stirred individual fractions were transferred with a Pasteur pipet into a Petroff-Hauser bacteria counting chamber and observed under phase contrast in a Zeiss RA microscope utilizing ~630 or ~1,000 magnification. The image was displayed on a viewing screen, and the widths and the lengths of individually isolated cardiac myocytes were measured with a ruler. Only cells that appeared intact, did not branch, and showed a distinct cross-striation were considered. The largest diameter of the cells was measured, and their length was estimated after averaging the irregularities of the intercalated discs at the discretion of the observer. In each cell the length and width were measured, their ratio calculated, and the volume determined on the assumption of a cylindrical configuration. The spontaneous contractions were of low frequency and the beating cells remained slack for about 10 s, which is enough for measurement. In each heart 100 cells were measured since, in a preliminary study, no significant differences were found among results obtained from 100, ZOO, 300, and 400 cells from the same heart. The average sarcomere lengths were estimated by direct measurement of a series of 20 sarcomeres on a clearly identifiable myofibril in one cell, and the mean value was then calculated. A total of 15-20 cells in each heart was investigated.
B. KORECKY
AND
K.
RAKUSAN
Cardiac hypertrophy was produced in normal male Sprague-Dawley rats (Bio-Breeding Laboratories), with body weights between 340 and 360 g, by constricting the abdominal aorta below the diaphragm with a silver ring. This technique has been described in detail elsewhere (7). A group of animals with a similar initial body weight was kept as controls. Both groups were sacrificed 2-3 wk after the operation, and the cardiac myocytes were isolated as above. To compare the ventricular weights (heart weights) of the two groups, each isolated and cannulated heart was trimmed of adhering tissues and submerged in Ringer solution. Then the stopper was removed, the remaining blood washed out by gentle massage, the liquid squeezed out of the heart, the stopper replaced, and the whole system weighted. After retrograde perfusion was completed the heart was transsected between the atria and ventricles, the cannula filled with liquid, and the original stopper reintroduced. The weight of the cannula with the atria was then subtracted from the original weight of the whole system, and the difference was taken as an estimate of the weight of the heart, excluding the atria, in normal animals and animals with aortic constriction. The morphometric analysis of the isolated cells was the same as above. RESULTS
The changes in dimensions of average left ventricular myocytes during the postnatal period between 1 and 8 mo of age in normal rats are summarized in Table 1. The average body weight increased approximately eightfold, from 81 g to 650 g Both cellular length and width increased by about 609& their ratio remaining remarkably constant through this period. The average cell volume increased from 14 x lO:j to 67 x 10” pm”, representing an almost fivefold increase. The morphometric data obtained from hearts of individual animals are displayed as a function of body weight in Fig. 1. If plotted on a double-logarithmic scale, the increases in width, length, and volume follow a straight line with respect to increasing body weight of the animals. Consequently, these relationships may be expressed by individual allometric equations, which wiI1 have a general form of y = 6x”, where y is the respective cell dimension, x is the body weight, b is a constant, and a is the coefficient expressing the slope 1. Body weights and cell dimensiorzs in five age groups of normal rats - -_---
TABLE
No. of FZats ~~--.
B&y
Wt, &
Cell
Length, tim ---- -.-.-. - --
Cell
Width, Pm
Cell Volume, pm” X 10:’
Length/Width --~
6
81.2k1.7
78.1H.3
14.9kO.5
5.3kO.11
14.421.2
9
159.8k7.4
84.8k2.1
16.7kO.4
5.19*0.06
19.7k1.4
9
365.9258
103.Od.O
20.550.2
5.14kO.06
35.621.0
463.5~12.1
119.922.2
22.2e0.3
5.48kO.10
48.4k2.1
647.8d3.0
127.5d.7
25.1kO.5
5.29&0.11
67.2k3.2
11 9 Values
are averages
t SEM.
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GROWTH
OF
28 24
CARDIAC
H125
MYOCYTE TABLE 2. Relationship between. cell dimensions and body weight calculated according to allometric equation ------------b tk r
- CELL WIDTH
20
____I”---^__--
0.253 Cell length, 24.49 Pm 4.78 0.251 Cell width, Pm 0.746 Cell volume, 0.47 pm;’ X 10:’ p-----__c____-Relationship between cell dimensions (y) grams, calculated according to the allometric constant b is the value of y when x is equal slope of the line, and its value indicates the to r; r = correlation coefficient; SEE = estimate.
80 70
- CELL VOLUME
0.942
0.0635
0.948
0.0596
0.961
0.1541
and body weight (x1 in equation: y = bx”. The to 1, constant cy is the growth ofy in relation standard error of the
TABLE 3. Body weights, heart weights, and cell dimensions in normal rats and in rats with aortic stenosis -~~_ -..-- -~. ----~-~. _.~
w3
x103
SEE
.----
20 15 10
B+
Wt,
-
a
~-. 75
100
150 200 300 400 BODY WEIGHT in g
600
FIG. 1, Mean values 295% confidence limits ( dashed lines) describing relationship between body weight and average dimensions of left ventricular myocytes in normal rats.
of’ the linear relationship and thus also the relative rate of growth with respect to body weight. The numerical values of the constants for the allometric equations are given in Table 2, The rate coefficients (a) for cell length and cell width are almost identical, which is additional evidence indicating a harmonic length-towidth relationship of normal growth of the cardiac myocytes. The high correlation coefficients and relatively small standard errors indicate a close relationship between the growth of the body and the growth of the basic morphological unit of the heart-the myocyte. In the case of stimulated cardiac growth observed after experimental aortic stenosis (Table 3), the average length, width, and volume of the left ventricular myocytes were significantly increased when compared to those of the normal control rats of similar body weight. As in the case of normal growth, there was no significant change in the length-to-width relationship. The above increments in cellular dimensions in hypertrophied hearts correspond to those that could be expected when the heart becomes larger due to normal growth, as illustrated in Fig. 2; When these average cell volumes are displayed on the growth chart of the normal cell volume-to-body weight relationship, we can see that cell volumes are higher in rats with aortic stenosis (hypertrophy) than in control rats of similar body weight. If we, however, compare the hypertrophied hearts with those of normal but heavier rats with the same heart weight (normal), the average volume of the myocytes appears to be the same. The relative frequency distribution of cell length, width, and volume of normal and hypertrophied hearts is disslaved in Fig. 3. These data are based on measure-
-..
Heart mR
Wt mg1100 IZBW
L-gth, *rn
---~-
Width, Pm
Len hl Wi $ th
Volume, pm” x
10"
--__
Controls (n = 11)
463.5 212.1
1143 k51
246 *5
119.9 k2.2
22.2 20.3
5.48 kO.10
48.4 k2.1
Stenosis (I?. = 11)
473.2 k 10.3
1381 229
293 +6
128.8 k2.4
25.1 LO.4
5.22 20.07
66.6 k3.2
SignifiNS coo1 coo1 1.02 coo1 NS -coo1 cance,--- ----P _ .~--~_ Values are averages ,+ SEM. BW = body weight, NS = not significant.
80
FIG. 2. Mean values +SEM of average volume of left ventricular myocytes in a group of control rats and rats with expFrimenta1 aortic stenosis (hypertrophy) plotted on growth chart expressing normal relationship between body weight and average volume of myocyte (see Fig. 1). Values for hypertrophic hearts are then extrapolated (dashed lines) to body weight, which would correspond to that of older normal rats having similar heart weight (normal).
ments of 100 cells in each heart and the values for individual classes were subsequently averaged for the whole group. It appears that in the case of hypertrophy, there is a general trend of increasing the size in all chosen myocytes, as represented by the arbitrarily classes.
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H126
B. KORECKY
RELATIVE
FREQUENCY
DISTRIBUTION
-COrJTRROL ---HYPERTROPHY
%
LENGTH
%
FIG. 3. Mean values of arbitrarily selected classes of cardiac myocytev with respect to their length, width, and volume in a group of control rats and rats after aortic constriction (hypertrophy). Volumes are expressed in units of pm x 10%
DISCUSSION
Enzymatic disruption of the left ventricular myocardium in the rat provides a yield of individual cells that shows morphological characteristics very similar to those observed in cells from” histological sections of the normal left ventricular myocardiumr The fact that these cells are isolated offers the advantage of inspection of the whole cell under the microscope. Approximately 50% of the observed cells exhibited normal morphology, while the remainder showed varying degrees of damage and were therefore not consider&d. since the final yield amounted to approxi mately half of the mass of the original tissue, we may assume that our sample of isolated cells contained ?O-30% of the original population of myocytes This relatively low yield could be considered adequate for further measurement, provided that the final sample may be shown to be representative of the original cell population of the left ventricle. We have no direct experimental proof that our sample is indeed representative, but some circumstantial evidence suggests that it may be so. First, no significant differences were found between momhometric measurements obtained from different fractions during the isolation procedure. Second, when samples were taken from different parts of the left ventricle after perfusion, no significant difference in morphometry of myocytes was observed. Third, when individual parts of the left ventricle were taken from one rat and the cells isolated by incubation in the enzyme solution with concurrent application of gentle shear stress (shaking or rotation), the morphometric data obtained after these isolation procedures did not significantly differ from those obtained after isolation by the retrograde perfusion technique. The selection of normal cells for measurement under the microscope depends on the decision of the observer, and the results may therefore be affected by an individual bias. All the presently reported data were those measured by one of us (B.K. ); measurements obtained in the same samples by other investigators in our
AND
K. RAKUSAN
laboratory did not differ significantly from those reported in this study. The average length of the sarcomeres after isolation, as determined concurrently in four to six hearts of each group, under the light microscope, was found to be 1.8 pm, which is approximately 0.1 pm less than was estimated for rat papillary muscle at slack length (4, 6) in different preparations. This degree of contracture is unfortunately unavoidable and occurs most probably during the enzymatic disruption of the heart with collagenase and hyaluronidase, which require CaZ+ for maintaining this activity, since, after addition of Ca buffer to the perfusing solution, the hearts could not be digested even after several hours of perfusion. Addition of EGTA (0.25 PM- 1 mM) to the final suspension of isolated cells did not abolish the existing partial contracture. Since the average sarcomere lengths, as measured under the light microscope, did not differ between myocytes of individual age groups, we may assume that this technique causes a constant degree of contracture which appears to be independent of the age and weight of the animal. The width of isolated adult cardiac myocytes (20-25 pm) appears to be greater than that reported by other investigators who utilized standard histological techniques (12-16 pm) (1, 2, 10, 12). This difference may be explained by a combination of the following factors: the isolated myocytes are in partial contracture and they are devoid of interstitium which may have restricted them. They probably exhibit a certain degree of swelling, since their sarcolemmas would be at least temporarily damaged during the separation from neighboring cells (e.g., cleavage of nexuses). All these factors would probably lead to increase of cell diameter. Since the cells have settled down in the chamber, it is likely that the greatest diameter will be the one which will yield itself for measurement. On the other hand, the preparation of myocardium for histology usually involves dehydration and subsequent shrinkage, which may lead to a decrease of cell diameter. The lengths of isolated adult cardiac cells are in the range of 100428 pm. We are not aware of any histological measurement of cell length in the rat but Laks et al. (8) reported an average value for adult dog cardiac myocytes of 71 pm. It seems therefore that both the width and the length of isolated myocytes are larger than those reported in histological preparations. It is likely that neither of the two techniques yields values that truly reflect the cellular size in vivo. Results based on histological techniques may be distorted by shrinkage, poor sampling, and deviation from cylindrical configuration of muscle cells. On the other hand, isolated myocytes may increase in size, due to absence of surrounding connective tissue and swelling. In addition, a certain selection could not be completely excluded because of the relatively low final yield. The only histological data dealing with postnatal increase of the width of cardiac myocytes in the rat (12) showed an increase from 6 pm at birth to 10 pm at 30 days and to 16 pm in adult animals. In the present study a similar relative increase (65%) in width was observed between the youngest group (ca. 30 days
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GROWTH
OF
CARDIAC
H127
MYOCYTE
old) and the oldest group, which was ea. 8 mo of age. The constancy of the relationship between length and width of cardiac myocytes during the above period indicates the harmonic growth of the cell, which, to our knowledge, has not previously been reported. Our data also indicate that the dimensions of myocytes increased in proportion to the increasing body weight over a range of 75-750 g. It would be tempting to calculate the total number of myocytes constituting the growing left ventricle by dividing its mass by the volume of its average myocyte and determine the total number of myocytes. Unfortunately, because of problems inherent in the technique of cell isolation, the left ventricular weight and the cell size could not be determined in the same heart. However the rate of growth of the mass of the left ventricle and the rate of growth of its myocytes may be compared indirectly taking into consideration the following. First, the average cell volume (V) may be related to the total body weight (BW) as V = 0,475 BW*=7”6 (see Table 2). Second, the left ventricular weight (LVW) may be also calculated as a product of average myocyte volume (V), total number of myocytes (NJ, and specific weight of cardiac tissue (D) divided by the fraction of the left ventricle formed by myocytes only (F); therefore, LVW = V x N x D/F. Finally, the directly determined left ventricular weight was found to be related to the total body weight according to the following allometric equation: LVW (mg) = 5.662 BW0v7-15, as determined previously in a large number of normal animals from our colony between 40 and 800 g in body weight. By comparing the values for the exponent Q! in the above two equations (0.746 and 0.745), we can conclude that the rates of growth of cell volume of the mass of the left ventricle were identical. If we assume a constant ratio between myocytes and interstitium and constant density (D/F = constant), the total number of myocytes constituting the left ventricle must have remained unchanged during the above age period. In case of stimulated cardiac growth, which was induced by increased afterload due to experimental aortic constriction, cellular hypertrophy was also observed by direct morphometric measurements from isolated myocytes. The larger cellular volume observed in this type of hypertrophy was similar to the volume of myocytes of normal rats of1 arger body weight but with heart weigh ts similar to the anim .als wi th experim ental cardiomegaly (see Fig. 2). Consequently, a simple cellular hypertrophy with no concomitant increase in the total number of myocytes would be the most probable explanation for the stimulated cardiac growth of adult rats as well.
However, it should be pointed out that the cardiac hypertrophy in our experimental group was of relatively mild degree: the final heart weight in animals with aortic constriction did not exceed the heart weight in normal animals with the highest body weight. Some of our results, especially those concerning the symmetrical cell growth may not necessarily apply to the cases of excessive cardiac hypertrophy and/or cardiac dilatation. Nevertheless, the conclusions that we derived from our results on normal and stimulated cardiac growth agree with those reported by others (5, 9, 13) who studied the normal and stimulated cardiac growth in humans by determining the density of cardiac muscle muclei in histological preparations from normal and hypertrophic hearts. From our present results we may conclude that normal cardiac growth in the rat fro& weaning to adulthood is almost entirely due to the growth of existing cells, i.e., cellular hypertrophy. In addition, the stimulated growth of the adult heart was also accomplished by accelerated growth of existing myocytes. There is, however, some experimental evidence that the proliferation of cardiac myocytes as a mode of heart growth is present during the first 3-4 wk of postnatal life in the rat (12, 13). During this period, the rate of growth of the heart is faster than later on, and in the case of the left ventricle, it may be related to the increase in body weight as LVW = 2.586 BW1.0g2 (11). Unfortunately, we have no morphometric data on cardisc myocytes for this age because of technical difficulties associated with their isolation. However, if we extrapolate the cell volume-to-body weight relationship established for older animals into this early postnatal period, then the rate of heart growth (a! = 1.092) becomes faster than the rate of cell volume growth (a = 0.745). Assuming an average body weight at birth of 5 g and at 3 wk of 40 g, a twofold increase in the total number of myocytes would be predicted to explain the normal increase in cardiac mass for the above period. The extrapolation of the cell volume-to-body weight relationship into this early postnatal period is speculative, but it is interesting to note that a similar increase of the total number of myocytes was postulated for the first 3 wk of postnatal life in the rat by Hort (5) in his morphometric studies based on evaluation of nuclear densities with respect to heart weight. This Medical
Received
study was supported by Ontario Research Council of Canada.
for publication
27 June
Heart
Foundation
and
1977.
REFERENCES
ARAI,
S., A. MACHIDA, AND T. NAKAMURA. Myocardial structure and vascularization of hypertrophied hearts. T&&u J. Exptl. Med. 95: 35-54, 1968. 2. ASHLEY, L. M. A determination of the diameters of ventricular myocardial fibers in man and other mammals. Am. J. Anat. 77: 325-364, 1945. 3. BERRY, M. N., D. S. FRIEND, AND J. SCHEUER. Morphology and metabolism in intact muscle cell isolated from adult rat heart. Circdation Res. 26: 679-687, 1970. 1.
4. GRIMM, A. F., K. V. KATELE, S. A. KLEIN, AND H. L. LIN. Growth of the rat heart. Left ventricular morphology and sarcomere lengths. Growth 37: 189-208, 1973. 5. HORT, W, Quantitative histologische Untersuchungen an wachsenden Herzen. Arch. Pathol. Anat. Physid. 323: 223-247, 1953. 6. JEWELL, B. R. A reexamination of the influence of muscle length on myocardial performance. Circutation Res. 40: 221230, 1977. 7. KORECKY, B., K. RAKUSAN, AND 0. POUPA. Experimental car-
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HE8 diomegaly. Methods Achiev. Exptl. PathoZ. 2: 158-171, 1967. 8. LAKS, M. M., M. J. NISENSON, AND H. J. SWAN. Myocardial cell and sarcomere lengths in the normal dog heart. Circulation Res. 21: 671-678, 1967. 9. LINZBACH, A. J. Mikrometrische und histologische Analyse hypertropher menschlicher Herzen. Arch. PathoZ. Amt. PhysioE. 314: 534-594, 19.17. 10. MUNELL, J. F., AND R. GETTY. Nuclear lobulation and amitotic division associated with increasing cell size in the aging canine myocardium. J. Gerontol. 23: 363-369, 1968.
B. KORECKY
AND
K.
RAKUSAN
11. RAKUSAN, K., B. KORECKY, 2. ROTH, AND 0. POUPA. Development of the ventricular weight of the rat heart with special reference to the early phases of postnatal ontogenesis. Physiol. Bohemoslov. 12: 518-524, 1963. 12. RAKUSAN, K., AND 0. POUPA. Changes in the diffusion distance in the rat heart muscle during development. Physiol. Bohemoslov. 12: 220-227, 1963. 13. ZAK, R, Cell proliferation during cardiac growth. Am. J. Cardial. 31: 211-219, 1973.
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