,J Mol Cell Cardiol

Nuclear

23, 833-839 (1991)

Size and DNA

A. Martin

Gerdes,

Content in Rat Cardiac MaturationandAging Martha C. Morales, Vandna andMarvin R. Ahmrez’

Myocytes

During

Handa,

Jo Ann Moore

Growth,

Department of Anatomy, University of South For&, Tampa, Florida 33612, USA and ‘Department of Biology, Universityof South Florida, Tampa, Florida 33620, USA (Received 1 October 1990, accepted in revisedfonn 28 January 1991) A. M. GERDES, M. C. MORALES, V. HANDA, J. A. MOORE AND M. R. ALVAREZ. Nuclear Size and DNA Content in Rat Cardiac Myocytes During Growth, Maturation and Aging. Jowmf ojM&c&~and Cell&r Cardiology (1991) 23, 833-839. Changes in nuclear volume and DNA content were examined in cardiac myocytes isolated from 21-day-old (wear&q, W), 3-month-old (adult, A), and P-year-old (old, 0) rats to document normal parameters for nuclear growth and DNA content. Nuclear volume was calculated from direct measurements of isolated myocyte nuclear profiles and DNA content was measured from DAPI-stained nuclei using an image analysis microdensitometry system. Myocyte volume was measured with a Coulter Channelyzer system. Nuclear volume increased 79% from W to A as a result of an increase in nuclear length. Nuclear width was unchanged. Nuclear volume was not changed from A to 0. Approximately 98% of the left ventricular myocytes from all three rat groups contained a diploid DNA content with the remainder of nuclei being tetraploid. The degree of polyploidy increased slightly, but significantly, in right ventricular myocytes from 0. Due to the substantially greater increase in myocyte volume relative to nuclear volume, nuclear volume percentage decreased from 3.65 * 0.28 to 1.64 * 0.13 from W to A but was unchanged from A to 0. To summarize: (1) nuclear volume of rat cardiac myocytes increases significantly during normal physiological growth (W to A) hut the rate of nuclear growth is less than that of cell volume; (2) the increase in nuclear size from W to A is not due to an increase in DNA content; (3) cardiac myocytes from Sprague-Dawley rats are predominantly diploid; and (4) there is little change in DNA content of cardiac myocytes from rats of this strain during growth, maturation and aging.

KEY WORDS:

Isolated

myocytes;

DNA

content;

Aging.

Introduction The majority of mammalian cells have a diploid DNA content. Some cells, however, are polyploid, possessing multiple amounts of the diploid DNA content. Hepatocytes and megakaryocytes are examples of cells that typically display polyploidy [3, 24, 261. Cardiac myocytes from human hearts also show increased degrees of polyploidy, beginning during childhood and increasing with age [I, 27, 28, 33, 3.51. Although the precise reason for the increase in cellular DNA is not clear, polyploidization of human cardiac myocytes is associated with aging and increased heart weight [27, 28, 331. The incidence of polyploidy in cardiac myocytes from rat hearts is not clear. Grove et al. [ 191 have indicated that most cells contain a Address for correspondence: 12901 N Bruce B. Downs, 0022-2828/91/070833

A. Martin Gerdes, Ph.D., Tampa, Florida 33612, USA.

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diploid DNA content while Stere and Anthony [29] have stated that 90% of rat cardiac myocyte nuclei are tetraploid. SpragueDawley rats were used in both studies. Engelmann et al. [14] reported that right ventricular and septal myocytes are largely polyploid while left ventricular myocytes are predominantly diploid in Wistar Kyoto (WKY) and Spontaneously Hypertensive rats (SHR). A major goal of this study was to examine DNA content of cardiac myocytes from Sprague-Dawley rats in an effort to clarify the extent of myocyte polyploidy in this rat strain. Other goals of these experiments were to determine: (1) if age-related changes in DNA content occur in cardiac myocytes from rats; and (2) if there are regional differences in DNA content of cardiac myocytes from this strain.

University

of South

Florida,

Department

of Anatomy,

@ 1991 Academic

Press

MDC Limited

6,

834

A.M.

Gel&s

To address these points, nuclear DNA content was measured in isolated cardiac myocytes prepared from weanling (3-week-old), adult (3-month-old), and old (2-year-old) SpragueDawley rats. Myocytes from the right and left ventricles were examined in the 2-year-olds to determine if regional differences existed. A final goal of this study was to evaluate possible changes in nuclear size during growth, maturation and aging. Myocyte nuclear dimensions have been measured from tissue sections previously [6, 101. Myocyte nuclear volume has also been estimated from tissue sections [5, S, 181 and isolated nuclear preparations [21]. In the present experiments, the dimensions of cardiac myocyte nuclei were measured using isolated myocytes tixed in a manner that does not alter cellular dimensions [ 161. The DNA-specific fluorochrome, DAPI , was used to visualize nuclei and enable easy determination of maximum nuclear dimensions.

Materials and Methods Ex@rbnentulanimals Female Sprague-Dawley rats were obtained from the Holtzman Company (Madison, WI). Cardiac myocytes were examined from rats of the following ages: (1) 3 weeks old; (2) 3 months old; and (3) 2 years old.

Cell isolationand volumemeasurements Isolated cardiac myocytes were prepared using a procedure outlined by Bishop and Drummond [II]. Rats were anaesthetized with sodium pentobarbital(45 mg/kg, i.p.) and the hearts were removed, blotted and weighed. The aorta was cannulated and attached to a modified Langendorff apparatus for retrograde perfusion of Joklik’s media followed by Joklik’s media containing collagenase (Worthington Biochemical Co, Freehold, NJ; 200 units activity/ml). Tissue was minced and cells filtered through nylon mesh (250 pm) into glutaraldehyde fixative (final concentration approximately 1.5 % in 0.08 M phosphate buffer) [ 14. A Coulter Channelyzer was used to determine the volume of fixed cells obtained from each heart. A detailed summary of this method has been published [17].

et al. Measurement of DNA contentandnuclearsize Isolated myocytes were stained with the DNADAPI (4’-6specific fluorochrome diamidino-2 phenylindole-HCl) for subsequent analysis of DNA content and nuclear size using a Bioquant Meg IV automatic image analysis system. Nuclear diameters and DNA content were measured from 200 nuclei from each sample. Myocyte nuclei were tagged by placing a drop of DAPI solution (a 100ml aqueous solution contains 1 mg DAPI, 138 mg Na3P04. 12H20, 0.9 g NaCl, 11 mg CaC12, 12 mg MgSO*- 7H20, 0.6ml Nonidet P-40) onto the glutaraldehyde-fored myocytes prior to adding the cover slip [31]. Chicken erythrocytes were used as a standard to determine the DNA content of cardiac myocyte nuclei in initial experiments. Mean DNA content for the primary peak on histograms of cardiac myocyte nuclei was approximately 6.3 pg. This agrees with reported values for DNA content of nuclei from rat heart [32, 341. The major and minor nuclear diameters were measured from DAPI-stained nuclei (random sample of mononucleated and binucleated cells) from isolated cell samples from each animal group. Nuclear volume was estimated from the following formula for a prolate ellipsoid

V= ?r/AB2

(1)

where V is the volume, A the long axis, and B, the short axis. A pilot study revealed that nuclei from individual binucleated cells had a similar volume and that nuclear volume was comparable in mononucleated and binucleated cells. Nuclear volume percentage (NVP) was determined from isolated myocytes in the following manner NVp=BF(2xNV)+MF(lxNV)

CV

(2)

where BF is the fraction of binucleated cells, MF the fraction of mononucleated cells, NV the nuclear volume [formula (1) above], and CV the cell volume (obtained with a Coulter Channelyzer). In each animal group, the percentage of binucleated cells was approximately 92 % (BF = 0.92), with the remaining cells being predominantly monunucleated

Analysis

of Rat Cardiac

(MF = 0.08). Since less than half of 1% of cells contained more than two nuclei, those cells were ignored. We reasoned that nuclear volume per cent (the percentage of myocyte cytoplasm occupied by nuclei) measurements from isolated cells should be similar to data obtained morphometrically from whole-sectioned tissue if the method for estimating nuclear volume was accurate. Therefore, myocyte nuclear volume percentage was also determined morphometrically from low-power electron micrographs of whole-sectioned tissue from 3-month-old adult rats. Nuclear volume percentage can be determined precisely with this method since nuclear boundaries are easily distinguished, percentage measurements are volume independent of sectioning angle, and nuclear diameters are much greater than section thickness [36]. Details of the procedures for tissue and morphometry have been processing described [ 15, 171.

ANOVA was used to compare data from 21-day-, 3-month-, and 2-year-old rats. The Scheffe test was used to examine significant differences observed with the ANOVA. Student’s t-test was used to compare left and right ventricular DNA content from old rats. ReSUltS The percentage of myocyte cytoplasm occupied by nuclei (myocyte nuclear volume percentage) was obtained from 3-month-old

TABLE

1. Body

weight,

heart

Bodyb weight

Hearth weight bd

kc) Weanling

weight

Myocyte

Nuclei

rats by two different methods. Morphometric evaluation of electron micrographs of wholesectioned tissue (n = 8) gave a value of 1.52 f 0.52 for nuclear volume per cent. A similar value of 1.64 f 0.13 was obtained from direct measurements of nuclear dimensions from isolated myocytes (n = 6, N.S.). Body weight, heart weight and left ventricular myocyte data from Pl-day(weanling), 3-month-(adult), and 2-year-old (old-age) rats are listed in Table 1. The ratio of heart weight to cell volume was the same for weanling (0.039), adult (0.039) and old-age rats (0.040). Nuclear length and calculated values for nuclear volume of isolated left ventricular myocytes increased significantly from 21 days to 3 months of age (Table 1). Nuclear width of isolated left ventricular myocytes was similar in all three groups. Values for nuclear length, width and volume were similar in myocytes from adult and old-age rats. A significant increase in cell volume was observed from 21 days to 3 months of age. A further increase was noted from 3 months to 2 years of age. Nuclear growth did not keep up with the increase in cytoplasmic volume observed from 21 days to 3 months of age. As a result, nuclear volume per cent declined as cell volume increased. Nuclear volume per cent was reduced from 3 months to 2 years of age but the change was not statistically significant. The percentages of diploid and polyploid cells are indicated in Table 2. The vast majority of cardiac myocytes at each age were diploid. Polyploid nuclei were almost exclusively tetraploid in each animal group. A

and left ventricular NucleaP length

Nuclear width

(a4

myocyte

data.a

Nudea+ volume

Cellb volume

(w-4

@m3)

(pm3)

56 *7

262 It 40

10.62 f 0.26

4.69 *0.14

124

Adult

323 f 11

1037 f 68

17.12 f 0.24

5.03 kO.12

Old

420 f 52

1279 f 37

16.34 zt 0.42

4.89 f 0.09

W