Altered Balance Between Cell Replication and Apoptosis in Hearts and Kidneys of Newborn SHR
Abstract Apoptosis is involved in neonatal remodeling of organs of the cardiovascular system. Since we previously reported hyperplasia of these organs at birth in several forms of genetic hypertension, the aim of this study was to determine whether alterations of the apoptotic process could explain our findings. The heart, aorta, and kidneys of newborn Wistar-Kyoto and spontaneously hypertensive rats were harvested 24 hours after an injection of [3H]thymidine. DNA was extracted to measure its specific activity (index of DNA synthesis) and DNA fragmentation as an estimation of apoptosis. All organs studied showed an increased weight to body weight ratio in spontaneously hypertensive rats. Twenty-four hours after birth, DNA synthesis in all organs of spontaneously hypertensive rats was comparable to that in normotensive rats. However, apoptosis was markedly decreased in the heart and kidneys of newborn spontaneously hypertensive rats compared with their normotensive controls. In the aorta, apoptosis was reduced, but not significantly. Calculation of a proliferation index (DNA synthesis/fragmentation) revealed a significant increase of heart proliferation, with a similar trend in the aorta and kidneys. In addition, we found a negative correlation between heart weight and DNA fragmentation. Although other factors may influence hyperplasia of the aorta, we propose that a reduction of apoptotic activity is responsible, at least in part, for heart and kidney hyperplasia in newborn spontaneously hypertensive rats.
Already at birth, spontaneously hypertensive rats (SHR) have increased heart, kidney, and aortic weight, corrected or not for body weight.1 2 3 This finding was also reported for the heart and kidneys in other models of genetically hypertensive rats, such as Lyon and Dunedin (GH) strains.4 The increased heart weight was ascribed to hyperplasia as there was a significant rise in DNA concentration (μg/mg of protein) and no decrease in DNA per mg of tissue, an index of hypertrophy.1 2 Although tentative explanations have been put forward, including hemodynamic growth stimuli, abnormal growth of cardiac muscle cells, increased circulating growth factors and different kinetics of postnatal adrenergic innervation, the reason for this early cardiomegaly remains unknown.
Apoptosis, or programmed cell death, is a physiological process that is believed to maintain tissue integrity by counterbalancing cellular replication.5 However, the balance between the two processes has been shown to be altered in several physiological and pathophysiological conditions. In the heart, exaggerated apoptosis has been observed following ischemia-reperfusion injuries,6 cardiac hypertrophy,7 cardiac overload8 and heart failure9 (see Reference 1010 for review). Programmed cell death thus seems to be involved in situations of heart remodeling during or after pathological processes. It is therefore logical to believe that apoptosis may also be involved in postnatal maturation of the heart and other tissues of the cardiovascular system, which must adapt to their new hemodynamic role. Accordingly, a recent study reported increased apoptotic activity in heart ventricles shortly after birth (day 1) that subsided within 2 weeks.11 The conduction system of the heart is also modeled by postnatal apoptosis.12 Similarly, in newborn lambs, the abdominal aorta undergoes remodeling that involves apoptosis.13 This process therefore seems to be an important determinant of mature tissue architecture in the cardiovascular system.
In the context of hypertension, apoptosis has been previously shown to be enhanced in target organs of adult (12 weeks) SHR,7 while older rats may present reduced activity in comparison to normotensive controls.14 15 Since there seems to be dysregulation of the apoptotic process in this model of hypertension, the aim of the present study was to determine the functional balance between cellular replication (DNA synthesis) and apoptosis in newborn rats genetically predisposed to hypertension, in an effort to define the role of apoptosis in hyperplasia observed in target organs of hypertension at birth.
Pregnant female Wistar-Kyoto (WKY) and SHR were obtained from Charles River (St-Constant, Quebec) and kept in our animal facilities for 2 weeks before they delivered. Two to 3 hours after delivery, newborns were injected with 0.66 μCi/g IP [methyl-3H]-thymidine (New England Nuclear, 35 Ci/mmol) in a volume of 12.5 μL/g and were returned to their respective mothers (2 to 3 per strain) for 24 hours. They were then weighed and killed by decapitation. The heart, kidneys, aorta, and liver were rapidly harvested and frozen in liquid nitrogen. Kidneys from the same animal were studied together and 4 aortas from different animals were pooled. The liver of several animals was also pooled to serve as a standard (see below). Eight hearts and pairs of kidneys, as well as 4 pools of 4 aortas were studied. All procedures were approved by the animal care committee of Hôtel-Dieu de Montréal.
DNA Extraction and Estimation of DNA Synthesis
Frozen tissues were weighed and pulverized in liquid nitrogen–dry ice with a mortar and pestle, and processed as described previously.8 16 Briefly, the powder was weighed and digested for 3 hours at 50°C in 500 μL lysis buffer (EDTA 20 mmol/L, Tris-HCl 50 mmol/L and SDS 0.5% w/v) containing Proteinase K (500 μg/mL, GIBCO BRL). The tissues were then incubated for 1 hour at 37°C with RNase (250 μg/mL, GIBCO BRL). Samples were extracted using a standard chloroform:isoamyl alcohol (24:1) and phenol procedure. DNA was precipitated with potassium acetate (1.4 mmol/L, pH 8.0) and cold 95% ethanol. It was recovered by centrifugation, washed twice with 75% ethanol, and dried in a Speedvac. The DNA was then resuspended in 50 μL of water, heated at 65°C for 1 hour, and 1 μL was diluted in 100 μL water for quantification by UV spectrophotometry (260 nm). Radioactivity was counted in 5 μg of DNA to determine the extent of [methyl-3H]-thymidine incorporation into DNA, an estimate of DNA synthesis.
Quantification of Apoptotic Activity
One microgram of DNA was labeled at its 3′ end with 1 μL of [32P]-dCTP (3000 Ci/mmol, Amersham) and 1 μL of terminal deoxynucleotidyl transferase (tdt, GIBCO BRL) for 60 minutes at 37°C.16 One microliter of a base pair marker (λDNA/HindIII fragments, GIBCO BRL) and 1 μg of liver DNA were labeled simultaneously. For each sample, increasing concentrations of DNA were loaded on 1.5% agarose gel: 0.05, 0.1, 0.2, and 0.4 μg. In addition, the base pair marker and DNA obtained from pooled livers (serving as an internal control) were loaded on each gel. After gel electrophoresis, the radioactivity was transferred to a Hybond-N+ membrane (Amersham), which was exposed for 36 hours in a PhosphorImager cassette. Average optical density (OD) was calculated for the following regions: 150 to 1500 bp (apoptosis type B) and 20 to 30 kbp (apoptosis type A, see Fig 1A⇓). As reported previously,8 16 the slope obtained from the average OD of 4 DNA concentrations was used to quantify apoptosis in each sample. This slope was then divided by the slope obtained for the liver on the same gel (Fig 1B⇓) in order to account for differences in labeling and radioactivity transfer between gels and to increase reproducibility.
Additional Estimation of Apoptosis
We took advantage of the fact that DNA was intensely labeled with [3H]-thymidine in these newborn rats to determine the extent of new cells (labeled DNA) that went to apoptosis within 24 hours. For each sample, 10 μg DNA was loaded on 1.5% low-melting agarose gel (containing 10 μg/mL ethidium bromide) along with a base pair marker. The bands were then cut under UV light in 3 sections: genomic DNA, apoptosis type A, and apoptosis type B fragments. Agarose was melted at 65°C, dissolved in 37°C scintillation liquid, and counted for radioactivity. Pilot studies with this method revealed that agarose did not interfere with radioactivity measurement and showed very good reproducibility. The data are presented as a percentage of radioactivity recovered in the fragments (type A and B added) over that found in genomic DNA. As for other results, the unpaired t test was used to compare differences in values obtained in WKY and SHR. P values inferior to .05 were considered to be significant.
The litter size of WKY and SHR was similar (8.0 and 7.3, respectively), as was weight of the neonates: 5.41±0.35 and 5.22±0.39 g, respectively. As shown in the Table⇓, the cardiovascular organs were heavier in SHR than in WKY neonates, and even more so when the values were corrected for body weight (Fig 2⇓).
Total organ DNA content was similar in the hearts and kidneys, but was significantly elevated in the SHR aorta (Table⇑). Similar results were obtained for the total radioactivity of labeled DNA ([3H]-DNA, Table⇑). When expressed per milligram of tissue, DNA content was not significantly different in all tissues studied (Table⇑), as was the specific activity of DNA (radioactivity of labeled DNA expressed per microgram of DNA).
The fragmentation indexes of both type A (P=.074) and type B (P<.05) apoptosis were reduced in SHR hearts (60% decrease of type A, 80% decrease of type B). In both kidneys and aorta, the values had a tendency to decline, but the difference did not reach statistical significance (Fig 3⇓). Since the implication of each type is still not clear,17 both type A and type B apoptosis were measured and showed a similar pattern of variation. In addition, there was a negative correlation between the heart/body weight ratio and the type B fragmentation index (r=.551, P=.032, n=16), suggesting that decreased apoptosis may be a determinant of neonate heart weight. Fig 4⇓ depicts the ratio between the specific activity of DNA (DNA synthesis) and fragmentation for apoptosis type B, as an expression of the balance between both processes. This figure highlights the fact that the hearts seem to be in a proliferative state, despite similar DNA synthesis, due to reduced apoptotic activity. A similar trend was observed in the aorta and kidney (NS).
Estimation of apoptosis by separation of genomic and fragmented DNA labeled with [3H]-thymidine, a method which allows the evaluation of a percentage of DNA fragmentation in each organ, yielded similar results as our standard method of quantification (Fig 5⇓). With this method, apoptosis was significantly reduced in the heart and kidneys of SHR. In addition, the data obtained with this method suggest that newly replicated cells are more prone to undergo apoptosis in the aorta than in the heart and kidney in both WKY and SHR (P<.05, ANOVA+Bonferroni).
The present study is the first account of reduced apoptotic activity in the heart and kidneys of neonatal rats genetically predisposed to hypertension. A similar dysfunction of the apoptotic process may also occur in the aorta. In physiological conditions, apoptotic activity has been reported to be elevated in cardiac myocytes (and preferentially in the right ventricle) from the first day after birth and gradually subsides within 2 weeks.11 In the context of hypertension, however, we now report a marked decrease of apoptotic activity during the neonatal period. This is in sharp contrast with the increased activity reported previously in the same organs in adulthood.7 Preliminary results in SHR suggest that there is a slow rise in apoptotic activity over time (from the first to the sixteenth week of age) in cardiovascular tissues, followed by a decline.14 The lower expression of programmed cell death observed early in life may be a determinant of hyperplasia1 2 4 and of the increased heart weight that is seen shortly after birth in genetically hypertensive animals. Indeed, since there was no change in DNA synthesis, reduced apoptosis favors cellular accumulation, as depicted in Fig 4⇑. Moreover, an inverse correlation was found between heart weight and apoptotic activity, again indicating a relationship between these two parameters. Postnatal apoptosis has been suggested to determine the geometry of the right ventricle, particularly during transition from the fetal to the newborn circulatory system,11 and to eliminate potential arrhythmogenic areas of the conduction system.12 Although the consequence of blunted apoptotic activity in the hearts of SHR is not known, it may contribute to the development of cardiac hypertrophy later in the disease process by failing to remove the appropriate amount of cells during the neonatal period. Indeed, we recently observed that neonatal heart weight is a significant predictor of adult heart weight in recombinant inbred strains.18
The lack of increase in cellular replication in the heart reported here is at variance with previous findings1 when we studied neonates 6 hours after birth and not at 24 hours as in the present investigation. In vitro, SHR vascular smooth muscle cells show more rapid entry into the S phase of the cell cycle, but no change in the number of cells entering the cycle.19 This may explain the apparent discrepancy between the results obtained at 6 and 24 hours in vivo, as increased cell proliferation may be more pronounced immediately after birth. In addition, a larger sample size may be needed to detect a significant difference.
The mechanism responsible for the marked decrease of apoptotic activity in the heart early after birth in SHR is not known. However, due to the early onset of this phenomenon, it does not appear to be secondary to the development of hypertension. The genetic determinants of this alteration are now being explored. Since cardiomyocytes have been shown to undergo apoptosis during postnatal maturation of the heart,11 and since we observed a marked reduction (80%) of apoptotic activity, the cell type affected in SHR hearts most probably includes myocytes, but this needs to be assessed.8 Indeed, decreased apoptosis of other cell types, such as fibroblasts or endothelial cells, may also contribute to the difference.
Apoptotic activity was also reduced in the kidney, as demonstrated by the proportion of fragmented DNA over genomic DNA labeled in vivo. This is in accordance with our previous observation of heart and kidney hyperplasia in newborn SHR1 2 and the organ weight increase seen in this study. In contrast to the heart and kidney, the aorta did not show a decrease of programmed cell death, despite its augmented weight. At 24 hours, we recorded only a trend of an altered balance between DNA synthesis and apoptosis in this tissue (Fig 4⇑). This more subtle alteration may contribute to the increase in organ mass, but it is also possible that in the aorta, hyperplasia is due to an early rise in proliferation, as reported previously 6 hours after birth.1 Indeed, the results suggest that cellular replication is exaggerated either during placental life or very early after birth.
Apoptosis is normally present in tissues showing cellular replication activity and is generally weaker in quiescent adult tissues.5 Our results depicted in Fig 5⇑, which represents the extent of apoptosis in vivo in newly replicated cells, indicate that in the newborn, many cells of the aorta undergo apoptosis shortly after DNA synthesis, while the heart and especially the kidney have less apoptotic activity in newly formed cells. This difference in apoptotic activity between organs may reflect heterogeneity in local adaptation to the new hemodynamic function brought by the transition from fetal to newborn circulation. This evaluation is, however, pertinent only to newly replicated cells, as apoptosis occurring in the “total pool” of cells measured by 3′-end labeling, cannot be related directly to total DNA by the method used here.
In conclusion, the heart, kidneys, and aorta of newborn SHR show an increased mass compared to age-matched WKY controls. We propose that decreased apoptotic activity contributes to the increased organ weight and hyperplasia observed at birth in this genetically hypertensive rat strain. The mechanism responsible for the low expression of apoptosis should be determined, as this alteration may contribute to the pathogenesis of cardiac hypertrophy and even hypertension. However, to distinguish a potential pathogenetic mechanism from a simple strain difference, this phenotype should be tested by means of other genetic approaches such as segregation analysis of F2 hybrids or analysis of recombinant inbred strains.18
This work was supported by a grant from the Medical Research Council of Canada (MRC) and from Bayer Canada. P.M. holds a fellowship and B.S.T. a studentship from the MRC. Fruitful scientific discussions with Drs Denis deBlois and Johanne Tremblay of the CHUM Research Centre are also acknowledged. The secretarial assistance of Josée Bédard-Baker and the editorial help of Ovid Da Silva are appreciated.
Reprint requests to Pavel Hamet, MD, PhD, Laboratory of Molecular Pathophysiology, Centre de Recherche–CHUM, Pavillon Hôtel-Dieu, 3850 St. Urbain St, Montréal, Québec H2W 1T8, Canada.
- Received March 18, 1997.
- Revision received April 30, 1997.
- Accepted May 20, 1997.
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