THE ANATOMICAL RECORD 226207-212 (1990)

Influence of Age, Growth, and Sex on Cardiac Myocyte Size and Number in Rats SHULING BAI, SCOTT E. CAMPBELL, JO A N N MOORE, MARTHA C. MORALES, AND A. MARTIN GERDES Department of Anatomy, College of Medicine, University of South Florida, Tampa, Florida (S.E.C., J.A.M., M.C.M., A.M.G.); Department of Anatomy, China Medical University, Shenyang, Peoples Republic of China (S.B.)

ABSTRACT The effects of altered neonatal nutrition on cardiac myocyte size and number was examined in 21-day-old and 3-month-old rats. Nutritional differences in growth rate were produced in newborns by adjusting litter size to four (fast-growing), eight (normally growing), or 16 (slow-growing)pups per litter. Isolated myocytes were prepared from animals in each group to evaluate changes in cell size and number. Heart weight (mg 2 S.D.), at 21 days of age, was 398 2 51 for “fast-growing’’rats, 329 43 for “normally growing” rats, and 228 k 24 for “slowgrowing” rats. Body weights showed a comparable decline with reduced nutrition. In adults, treatment-related differences in body and heart weight were present in males but not females. “Slow-growing’’rats had 21% fewer myocytes than “fast-growing’’rats at 21 days of age, a change that persisted in adults. Values for myocyte number from “normally growing’’ rats were intermediate between those of “fast and slow-growing’’ rats at both 21 days and 3 months of age. In each heart region of weanling rats, myocyte length and volume were smallest in 16 per litter rats. Cellular dimensions increased progressively with better nutrition. In adults, differences in myocyte size as a function of altered neonatal nutrition were not statistically significant. Sex-related differences in myocyte size or number were not found in males and females from any group a t the time of weaning. In adults, it was also noted that males and females have a similar number of cardiac myocytes, but the larger heart weight found in males was primarily due to increased myocyte size. Myocyte size and number were also examined in 2-year-old female rats. There were no differences in cardiac myocyte size or number between 2-year-old females and 8-monthold weight-matched females.

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Currently available data regarding changes in myoHollenberg et al. (1977) and Rakusan et al. (1978) examined the effects of litter size on early postnatal cyte size and number in aging rats are conflicting. We growth of the heart. Both studies indicated that 21- had access to a colony of 2-year-old female rats and day-old rats with increased nutrition (small litter size) have included myocyte size and number data from had more cardiac myocytes when compared t o 21-day- those animals in this communication. old rats with poor nutrition (large litter size). Our reMATERIALS AND METHODS sults showed comparable changes in myocyte number Experimental Models in similarly treated weanling rats. Neither Hollenberg Growth rate was altered in newborn rats (less than 1 et al. (1977) nor Rakusan et al. (1978) examined the long-term effects of altered postnatal nutrition on car- day old) by adjusting the litter size to either four, eight, diac myocytes. As a result, several important questions or 16 pups per litter. Animals were randomized by comremain unanswered. Are the effects permanent? Does bining the offspring from four or more mothers who improved nutrition actually stimulate myocyte hyper- delivered on the same day. Either four, eight, or 16 plasia or simply allow full expression of growth poten- pups were arbitrarily returned to a given mother. tial? In an attempt to answer these questions, differ- Adoption was facilitated by rubbing ether on the back ences in cardiac myocyte size and number were of each pup t o mask its scent. At 21 days and 3 months compared in adult animals reared in litters of four, of age isolated myocytes were prepared from rats from eight, or 16 pups. Another goal of this study was to examine sex-related differences in cardiac myocyte size and number. Received August 26, 1988; accepted May 17, 1989. Animals from the above-mentioned nutritional study Address reprint requests to A. Martin Gerdes, Ph.D., University of were subdivided into males and females for this pur- South Florida, Department of Anatomy, Box 6, 12901 N. 30th Street, Tampa, FL 33612. pose. 0 1990 WILEY-LISS. INC

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each group. Sex-related differences were also examined in each animal group. The effects of aging on myocardial cell size and number was examined in 2-year-old females. These rats were compared to younger (8-month-old), weightmatched females. All rats used in these experiments were of the Sprague-Dawley strain obtained from Holtzman Company (Madison, WI). Myocyte Isolation and Morphometry

from each isolated cell preparation to obtain regional heart weight. Myocyte volume percentage was calculated morphometrically from perfusion-fixed tissue sections obtained from weanling rats. Since work by Anversa e t al. (1986) indicated that myocyte volume fraction does not change with aging (despite a slight increase in connective tissue), values from myocyte volume fraction obtained previously from adult rats (Gerdes et al., 1986) were used to calculate cell number in aging and adult rats. Myocyte volume percentage for whole-tissue, rather than contractile tissue alone (areas containing capillaries and myocytes but devoid of large blood vessels and connective tissue), was used to obtain more reliable values for cell number (Gerdes et al., 1986).

Rats were anesthetized with a n intraperitoneal injection of sodium pentobarbital (50 mglkg), and hearts were quickly removed, trimmed of excess tissue and fat, blotted, and weighed. The aorta was cannulated for retrograde coronary perfusion with calcium-free Joklik media containing 0.1 mM ethyleneglycol-bis-(B-amiStatistical Analyses noethyl ether) N,N’-tetraacetic acid (EGTA), which Student’s t test for unmatched pairs was used to comwas followed by Joklik media with collagenase. This procedure has been described previously (Gerdes et al., pare heart weight, heart weightlbody weight ratio, and myocyte volume and length measurements from 21986). Following collagenase treatment, hearts were di- year-old females and weight-matched younger females vided into regions using sharp dissection. In the litter (8 months old). Student’s t test was also used to comvariation study, hearts were divided into right ventric- pare data from males and females within a given treatular free wall, septum, and left ventricular free wall. ment group. In the litter size experiments, analysis of Isolated myocytes were collected only from the left and variance was used for comparisons. When significant right ventricular free wall in 2-year-old rats. The col- differences were indicated by one-way analysis of varilected tissue was minced in calcium-free, EGTA-con- ance, multiple comparisons were made using Scheffe’s taining Joklik media and poured through nylon mesh Method (Wallenstein e t al., 1980). Median values for (250 pm). Freshly isolated cells were fixed immediately cell volume and mean values for cell length were used in 1.5% glutaraldehyde in 0.08 M phosphate buffer when comparing a given region of experimental groups. (Gerdes et al., 1982). Cell volume of isolated myocytes was determined usRESULTS ing a Coulter Channelyzer (Model C256) linked to a Changes in four, eight, and 16 per litter rats a t 21 Coulter Counter (Model ZBI). The Coulter system determines cell volume by measuring the change in elec- days and 3 months of age are reported in Figures 1-4. trical resistance across a n aperture resulting from the The number of animals in each experimental group is displacement of electrolyte a s cells move through the given in Table 1. At 21 days of age, heart and body aperture (Nash et al., 1979; Gerdes et al., 1986). Based weight were inversely related to litter size (Fig. 1). In on the work of Hurley (1970), a shape factor of 1.05, adults, treatment-related differences in heart and body representing a cell lengthlwidth ratio of approximately weight were present in males but not females (Fig. 1). At 21 days of age, cell volumes increased as nutri7, was used. Cell length, defined as the longest length parallel to tional levels increased (Fig. 2). The same trend was the longitudinal axis of the myocyte, was measured noted in each region of the heart. In adults, myocyte directly using a phase-contrast microscope. Thirty-five volume tended to be larger in males with increased myocytes from each region of each animal were mea- levels of neonatal nutrition (Fig. 2). Adult females did not display any clear trends regarding changes in myosured. The number of nuclei per myocyte was counted from cyte volume. Cell length increased with augmented nutritional a sample of 200 isolated cells from each region of each animal group. Nuclei were counted using fluorescence levels in 21-day-old rats (Fig. 3). The same trend was microscopy after staining glutaraldehyde-fixed myo- noted in each region of the heart. In adult males and cytes with 4‘-6-diamidino-2 phenylindole-2 HC1 females, there were no differences in cell length among (DAPI). any treatment groups (Fig. 3). Regional weights were obtained from freshly excised Cell Number Determination hearts from anesthetized weanling and adult rats. This The procedure for calculating myocyte number has data was used to calculate cell number. In 21-day-old been outlined in detail elsewhere (Gerdes et al., 1986; rats, the relative percentage of whole heart was 85.6 f Campbell and Gerdes, 1987). Briefly, myocyte number 2.0% for ventricles, 66.5 f 2.9% for left ventricle plus was calculated for a given heart region by dividing the septum, 47.9 f 3.2% for left ventricular free wall, 19.1 product of myocyte volume fraction and tissue volume 2 2.7% for right ventricular free wall, 18.6 k 2.8% for of that region by the average myocyte volume. Re- septum, and 14.4 2 2.0% for atria. In adult rats, the gional weight divided by specific gravity gives tissue relative percentage of whole heart was 92.0 2 1.0% for volume. Since regional heart weights could not be ac- ventricles, 72.3 f 1.8% for left ventricle plus septum, curately obtained from collagenase-perfused hearts, 47.1 5 3.2% for left ventricular free wall, 19.7 t 1.7% relative percentage of a given region was obtained from for right ventricular free wall, 25.2 k 3.3% for septum, freshly excised hearts and multiplied by heart weight and 8.0 f 1.1% for atria. There were no sex- or nutri-

CARDIAC MYOCYTE SIZE

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tion-related differences in relative values for regional heart weights. Myocyte volume percentage, calculated morphometrically, was approximately 74% in both left and right ventricles from weanling and adult rats. Although the number of myocytes from each ventricular region was calculated for each animal group, we decided to report only the combined ventricular data (left and right ventricle plus septum) for purposes of simplification. This was deemed appropriate since regional alterations in myocyte number indicated the same general changes.

Poor nutrition reduced myocyte number by a similar degree in weanling and adult “slow-growing” rats when compared to age-matched “fast-growing” rats (Fig. 4).Due largely to individual variations in myocyte number, comparisons among treatment groups of weanling and adult rats were either marginally significant or insignificant (P values ranged from .03 to .08). Heart weight, body weight, and cell size and number were also compared between males and females from each treatment group at both ages. At the time of weaning, there were no significant differences in heart

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TABLE 1. Number of Animals

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CELL LENGTH ( p m ) Fig. 3.Alterations in cell length for 21-day-old and 3-month-old male and female rats from each group. Values are means -C S.D. in pm. Significant diffferences of P < .05 are indicated.

weight, body weight, and cellular dimensions between less of age, sex, or experimental treatment. The mean males and females from a given treatment group (cell percentage of binucleated cells ranged from 92% to size comparisons between male and female weanling 95%. Most of the remaining cells were mononucleated. rats are not shown). Within any given treatment Trinucleated and tetranucleated cells accounted for group, 3-month-old males had significantly larger body less than 1%of the myocytes in each animal group. weights and heart weights than 3-month-old females. DISCUSSION Compared to females, myocyte volume was signifiAlteration of Litter Size cantly greater in each region of each treatment group The effects of altered nutrition on the growth re(P < .001 for each comparison, Fig. 2). Adult males also had significantly longer myocytes in each cardiac re- sponse in neonatal rats has been examined by Hollengion than females (P < .05 for each comparison, Fig. 3). berg et al. (1977) and Rakusan et al. (1978). Results There were no significant differences in myocyte num- from both studies indicated that a t the time of weaning ber between males and females within a given treat- (21 days of age), rats from large litters (slow-growing rats) had smaller heart and body weights than rats ment group a t either age examined (Fig. 4). Two-year-old female rats weighed 409 -t- 49 g and from small litters (fast-growing rats). Hollenberg et al. had a mean heart weight of 1,247 69 mg (n = 8). (1977) noted reduced DNA labeling of myocytes, fibroThese rats were compared to younger 8-month-old, n = blasts, and endothelial cells in 21-day-old “slow8) females of a similar body weight (416 ? 14 g). There growing” rats. Using histometric methods, Rakusan et were no significant differences in heart weightibody al. (1978) found that “slow-growing” rats had 24% weight ratio (young, 2.85 0.25; old, 3.08 0.301, ven- fewer left ventricular myocytes than “fast-growing’’ tricular myocyte volume and length, or total number of rats. Our finding that “slow-growing” rats had approxventricular myocytes (young, 27.5 2.9 million; old, imately 21% fewer myocytes than “fast-growing” rats 28.4 ? 3.1 million). confirms the work of Rakusan e t al. (1978) and HollenThe percentage of mononucleated and binucleated berg et al. (1977). It seems clear from our data, howcells was similar in all regions of each group, regard- ever, that quantitating changes in myocyte number of

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CARDIAC MYOCYTE SIZE

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CELL NUMBER (x 106) Fig. 4. Changes in myocyte number for 21-day-old and 3-month-old males and females from each experimental group. All values are means C S.D. Significant differences of P < .05 are indicated.

such a small magnitude (e.g., 20% or less) is complicated by the inherent variation in cell number encountered in the normal population (nutrition-related changes in cell number were either marginally significant or insignificant for a given comparison). The long-term effects of litter size variation in adult offspring were not examined in the studies by Rakusan e t al. (1978) or Hollenberg et al. (1977). We have extended their work by examining cardiac myocytes from adult rats reared at four, eight, and 16 pups per litter. Our finding that myocyte number remained relatively stable in each group of rats from age 21 days to 3 months suggests that permanent changes in the myocyte population had occurred. It appears that poor neonatal nutrition leads to adult hearts with fewer cardiac myocytes. It is also apparent that slow-growing rats given free access to rat chow a t the time of weaning were not able to compensate for the altered myocyte number with a period of hyperplasia. Clubb et al. (1987) have demonstrated that myocyte proliferation ceases shortly after birth. It is not clear whether nutritional deficiency resulted in cell death or simply slowed myocyte proliferation during the critical early neonatal period. Based on the work of Widdowson and Kennedy (1962), it appears that the alterations observed here as a result of neonatal nutrition result in permanent changes. Reduced neonatal nutrition appears to have a more dramatic long-term effect on body and heart weight in males than in females. This may be related to interstitial cell damage and altered testosterone metabolism, which is known to occur in male pups raised in large litters (Widdowson et al., 1964). Sex- and Age-Related Differences in Myocytes

It is well known that male rats attain a larger body and heart weight than female rats (Addis and Gray,

1950). Sex-related differences in myocyte size and number, however, have not been adequately documented. In 3-month-old adult rats, we found that males have a similar number of larger myocytes than females. Based on total ventricular DNA content, Koenig et al. (1982) also concluded t h a t cell size, rather than cell number differences, is responsible for the larger heart weights found in males. It appears that the altered growth response in male and female rats is due to sex hormones (Koenig et al. 1982; Slob and Bosch, 1975). Although i t is interesting to speculate, i t is not known if the larger myocyte size contributes to the increased cardiovascular risk found in males. Age-related ultrastructural changes in myocardial cells have been documented in rats (Travis and Travis, 1972; Tomanek and Karlsson, 1973; Sachs et al., 1978). There are, however, relatively few reports on changes in cardiac myocyte number in senescent rats. Anversa et al. (1986) noted a n 18% loss of left ventricular myocytes in 20-month-old male SpragueDawley rats. The number of right ventricular myocytes was not changed. Myocyte hypertrophy was present in left but not right ventricles. Rakusan et al. (1984) found that left ventricular myocyte number was not altered in 23-month-old male spontaneously hypertensive and normotensive rats. In agreement with the findings of Rakusan et al. (19841, myocyte number was unchanged in 2-year-old female SpragueDawley rats used in this study. When compared to younger weight-matched rats, there was no myocyte hypertrophy in old animals. It is possible that male and female Sprague-Dawley rats respond differently to aging. Methodological differences may also account for the discrepancy between our data and that of Anversa et al. (1986). In conclusion, it was shown that 1) reduced neonatal nutrition caused significant changes in cardiac growth;

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2) some of the observed cardiac changes related to poor neonatal nutrition appear to be permanent; 3) poor neonatal nutrition had a more pronounced long-term effect on males; 4) in adult rats, males have a similar number of larger myocytes than females; and 5) aging did not significantly alter myocyte size or number in female rats. Potential Problems

The reliability of the cell sizing methods used in this study have been extensively documented (Gerdes et al., 1986) and applied to many experimental situations with extremely consistent results (Clubb et al., 1987; Gerdes et al., 1987, 1988; Smith and Bishop, 1985; Smith et al., 1988). The Coulter Channelyzer dramatically accelerates the process of measuring myocyte volume, and a s a result we have collected data from several hundred rats in the past few years. We have obtained very consistent cell volume data from weanling and adult rats with a 200 km aperture tube on our Coulter Counter. Although the model C256 Channelyzer employed in our laboratory is linear, it is a good idea to calibrate with microspheres that are similar to the volume of cells being examined. This point is particularly true when examining small cells from young animals. Significant morphological differences in hearts from rats of the same strain but obtained from different suppliers were recently reported (Campbell and Gerdes, 1987). Occasionally, we have also observed differences in myocyte size and number among various shipments of a given strain of animals from the same facility (Gerdes, unpublished results). It is probably a good idea to request rats from the same colony at a given animal facility in order to obtain more consistent data. Ideally, one should always collect data from a similar number of control and experimental animals a t the same time. Increased mortality from experimental manipulation, technical problems, and other factors may, however, make this a difficult goal. ACKNOWLEDGMENTS

This work was supported by grant R01 HL30696 from the National Institutes of Health. We are grateful to Ms. Martha Ward for her support and assistance. LITERATURE CITED Addis, T., and H. Gray 1950 Body size and organ weight. Growth, 14:49-80. Anversa, P., B. Hiler, R. Ricci, G. Guideri, and G. Olivetti 1986 Myo-

cyte cell loss and myocyte hypertrophy in the aging rat heart. J. Am. Coll. Cardiol., 8:1441-1448. Campbell, S.E., and A.M. Gerdes 1987 Regional differences in cardiac myocyte dimensions and number in-Sprague-Dawley rats from different suppliers. Proc. Soc. Exp. Biol. Med., 186:211-217. Clubb, F.J., P.D. Bell, J.D. Kriseman, and S.P. Bishop 1987 Myocardial cell growth and blood pressure development in neonatal spontaneously hypertensive rats. Lab. Invest., 56:189-197. Gerdes, A.M., J. Kriseman, and S.P. Bishop 1982 Morphometric study of cardiac muscle. The problem of tissue shrinkage. Lab. Invest., 46:271-274. Gerdes, A.M., J.A. Moore, J.M. Hines, P.A. Kirkland, and S.P. Bishop 1986 Regional differences in myocyte size in normal rat heart. Anat. Rec., 215t420-426. Gerdes, A.M., J.A. Moore, and J.M. Hines 1987 Regional changes in myocyte size and number in propranolol-treated hyperthyroid rats. Lab. Invest., 57:708-713. Gerdes, A.M., S.E. Campbell, and D.R. Hilbelink 1988 Structural remodeling of cardiac myocytes in rats with arteriovenous fistulas. Lab. Invest., 59:857-861. Hollenberg, M., N.Honbo, and A.J. Samorodin 1977 Cardiac cellular responses to altered nutrition in the neonatal rat. Am. J . Physiol., 233:H356-H360. Hurley, J. 1970 Sizing particles with a Coulter counter. Biophys. J., 10:74-79. Koenig, H., A. Goldstone, and C.Y. Lu 1982 Testosterone-mediated sexual dimorphism of the rodent heart. Ventricular lysosomes, mitochondria, and cell growth are modulated by androgens. Circ. Res., 5Ot782-787. Nash, G.B., P.E.R. Tatham, P.T. Powell, V.W. Twist, R.D. Speller, and L.T. Loverock 1979 Size measurements of isolated rat heart cells using Coulter analysis and light scatter flow cytometry. Biochim. Biophys. Acta, 587:99-111. Rakusan, K., S. Raman, R. Layberry, and B. Korecky 1978 The influence of aging and growth on the postnatal development of cardiac muscle in rats. Circ. Res., 42:212-218. Rakusan, K., P.W. Hrdina, Z. Turek, E.G. Lakatta, H.A. Spurgeon, and G.D. Wolford 1984 Cell size and capillary supply of the hypertensive rat heart: Quantitative study. Basic Res. Cardiol., 79: 389-395. Sachs, H.G., J . Colgan, and M.L. Lazarus 1978 Ultrastructure of the aging myocardium: A morphometric approach. Am. J . Anat., 150: 63-72. Slob, A.K., J.J.V.D.W.T. Bosch 1975 Sex differences in body growth in the rat. Physiol. Behav., 14t353-361. Smith, S.H., and S.P. Bishop 1985 Regional myocyte size in compensated right ventricular hypertrophy in the ferret. J . Mol. Cell. Cardiol., 17:1005-1011. Smith, S.H., M. McCaslin, C. Sreenan, and S.P. Bishop 1988 Regional myocyte size in two-kidney, one clip renal hypertension. J. Mol. Cell. Cardiol., 20:1035-1042. Tomanek, R.J., and U.L. Karlsson 1973 Myocardial ultrastructure of young and senescent rats. J . Ultrastruct. Res., 42:201-220. Travis, D.F., and A. Travis 1972 Ultrastructural changes in the left ventricular rat myocardial cells with age. J. Ultrastruct. Res., 39:124-148. Wallenstein, S., C.L. Zucker, and J.L. Fleiss 1980 Some statistical methods useful in circulation research. Circ. Res., 47:l-9. .Widdowson, E.M., and G.C. Kennedy 1962 Rate of growth, mature weight and life-span. Proc. R. Soc. Lond. [Biol.], 156:96-108. Widdowson, E.M., W.O. Mavor, and R.A. McCance 1964 The effect of undernutrition and rehabilitation on the development of the reproductive organs: Rats. J . Endocrinol., 29:119-126.

Influence of age, growth, and sex on cardiac myocyte size and number in rats.

The effects of altered neonatal nutrition on cardiac myocyte size and number was examined in 21-day-old and 3-month-old rats. Nutritional differences ...
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