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REGULATION IN GENETICALLY MODIFIED ANIMALS
Reynolds Center on Aging, Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Submitted 11 October 2002 ; accepted in final form 23 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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16% in the hearts of mice during adult
aging. To model the effect of mild SRF elevation in the aging heart,
transgenic mice with low levels of SRF overexpression were generated. By 6 mo
of age, the transgenic mice had a 19% increase of heart-to-body weight ratio
compared with nontransgenic mice. In addition, they had a 12% increase in
myocyte size, a 6.7% increase in collagen deposition, and altered gene
expression of a number of muscle-specific and cardiac genes. Doppler
echocardiography revealed that these transgenic mice had increased left
ventricular wall thickness and decreased left ventricular (LV) volumes,
increased LV stiffness with 20% reduction in early diastolic LV filling (peak
E), and 35% decline in peak E-to-peak A (late diastolic filling) ratio. The
observed changes, especially those in the E/A ratio, are similar to those seen
clinically in late life as a part of human adult myocardial aging. transcription factor; transgenic; cardiac structure and function
16-20%.
In addition, the mRNA levels of a number of SRF target genes, such as atrial
natriuretic factor (ANF), skeletal 
-actin, cardiac -actin,
-myosin heavy chain (-MHC), and 
-MHC, have been reported
to undergo changes during maturational development and growth, cardiac
hypertrophy, and in some instances cardiomyopathy
(3,
11,
12,
14). These findings suggest
that SRF may also play an important role in the regulation of genes that are
responsible for the maintenance of cardiac structure and function
(56).
In previous studies, we observed that SRF binding activity to its cognate
response sequence, the SRE, of the c-fos promoter appeared to be slightly
increased in the hearts of old rats compared with young adult rats
(47). Furthermore, the basal
expression of SRF protein was increased by 20% in the hearts of old rats
compared with young adult animals
(28,
52). To pursue the
significance of the increased SRF and its potential contribution to cardiac
changes during aging, we previously generated transgenic mice with moderate to
high levels (at least 1-fold increase compared with nontransgenic littermates)
of cardiac SRF overexpression
(56). These transgenic mice
developed enlarged hearts with cardiomyopathy, and all died within 6 mo after
birth. Also, the results suggested that moderately high levels of SRF
transgene overexpression correlated with earlier onset of cardiomyopathy and
earlier mortality in a dose-dependent manner. However, it was unclear whether
a mild increase of 20%, such as is observed during normal adult aging in the
rats, or a 16% increase as observed in mice would have any effect. We
therefore decided to generate transgenic mice with mild overexpression of SRF
in the heart to better mimic the normal aging process. Using a previously
tested DNA construct (56), we
produced transgenic mouse lines with mild cardiac overexpression of SRF of
49%.
Interestingly, there were cardiac changes in the apparently healthy young
adult transgenic mice at 6 mo of age that resembled those that usually occur
much later (18-22 mo) during the aging process in the old mice hearts and
mirrored those which have often been observed clinically in elderly
individuals.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-MHC promoter (a generous gift from Dr. J. Robbins,
The Children's Hospital and Research Foundation, Cincinnati, OH) and human SRF
cDNA (a generous gift from Dr. R. Prywes, Columbia University, New York, NY)
was constructed. It was linearized and was injected into the pronuclear stage
zygotes of FVB/N mouse strain according to the standard transgenic procedure
of Beth Israel Deaconess Medical Center transgenic facility. At 2-3 wk of age,
all animals had a 1-cm portion of tail removed for DNA analysis. The potential
transgenic mice were screened twice by the polymerase chain reaction using two
different forward primers (5'-ACAGGTGGTGAACCTGGACAC-3' and
5'-CCATTCAAGTGCACCAGGC-3') and one reverse primer
(5'-CACTGGAGTGGCAACTTCCAG-3'). Southern blot analysis, using a
[-32P]dCTP-labeled SRF cDNA fragment from plasmid pCGNSRF,
was employed to confirm the identification of transgenic founder mice and to
determine the transgene copy number in the transgenic mice. The studies were
conducted with approval of the Institutional Review Board and are in
accordance with the Guiding Principles for Research Involving Animals and
Human Beings of the American Physiological Society. In all experiments
that were performed in this study, age- and sex-matched nontransgenic
littermates were used for comparison with the SRF transgenic mice.
Northern blot analysis. Total RNA was isolated from ventricular
tissue using the ULTRA-SPEC RNA isolation reagent (Biotecx Laboratories,
Houston, TX). Ten micrograms of total RNA was then fractionated on a 1%
formaldehyde-agarose gel and transferred to a nylon membrane (Amersham Life
Science) by capillary action in high-salt solution (10x SSC-1 mM EDTA).
Blots were prehybridized in a hybridization solution containing 7% SDS, 0.5 M
NaHPO4 (pH 7.2), and 200 mg/ml salmon sperm DNA for 5 h at 65°C and
followed by overnight hybridization with [
-32P]ATP-labeled
oligonucleotide probes or [-32P]dCTP-labeled SRF cDNA probe.
Blots were washed three times in 2x SSC-0.2% SDS at room temperature for
30 min and then in 0.5x SSC-0.2% SDS at 65°C for 15-30 min before
exposure to X-ray film.
The sequences of the oligonucleotide probes were as follows: ANF,
5'-CCGGAAGCTGTTGCAGCCTAGTCCACTCTGGGCTCCAATCCTGTCAATCCTACCCCCGAAGCAGCTGGA-3';
skeletal -actin,
5'-TGGAGCAAAACAGAATGGCTGGCTTTAATGCTTCAAGTTTTCCATTTCCTTTCCACAGGG-3';
sarcoplasmic reticulum Ca2+-ATPase (SERCA2),
5'-TCAGTCATGCAGAGGGCTGGTAGATGTGTTGC-TAACAACGCACATGCACGCACCCGAACA-3'.
A double-stranded SRF cDNA fragment from plasmid pCGNSRF was used as a probe to examine the mRNA level of SRF.
Histological analysis. After animals were killed, mouse hearts were immediately removed and placed in relaxing buffer (25 mM KCl in PBS). After treatment with relaxing buffer, the hearts were placed in 10% neutral-buffered formalin overnight. After fixing, the samples were subjected to a dehydration series and embedded in paraffin.
The atria were separated from the ventricles and then each ventricle was sectioned in 3- to 4-µm intervals from the apex upward. The sections were stained using standard hematoxylin and eosin (HE) or Masson Trichrome staining (MTS) protocols (Poly Scientific; Bayshore, NY). Photomicro-graphs were obtained using a Nikon ES400 microscope.
Measurement of wall thickness, myocyte size, and fibrotic area. Slides of cross sections of the heart taken at the level of the left ventricular (LV) papillary muscles were stained with HE or MTS. Ventricular wall thicknesses were measured under the microscope using an objective micrometer with 0.01-mm ruler markings. The septum, LV, and right ventricular (RV) free walls were each measured at the level of the papillary muscles, and the average thickness was reported as means ± SD in millimeters.
Measurement of cardiac myocyte size. Digital images of HE-stained sections of the heart were acquired at x400 magnification with a Polaroid digital microscope camera mounted on a light microscope (Nikon). True-color image analysis was performed using Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD). The slides were analyzed by two independent observers blind to the transgenic status of the mice. Ten fields from each section of the heart, LV free wall, septum, and RV free wall were chosen at random. Both endocardium and epicardial regions were included in the selection. In each one of the 10 fields, 100 cardiomyocytes with nuclear profiles were measured. Myocyte diameter was expressed as means ± SD in micrometers.
To evaluate the fibrosis in the mouse myocardium, the digital images of MTS-stained cross section of the heart were captured at x200 magnification. The true-color image analysis was performed by using the above software to quantify the collagen deposition as an indicator of fibrosis in the transgenic mice relative to that of the nontransgenic control mice. The observer performing the evaluations was blind to the transgenic status of the mice. The fibrotic areas stained blue with the MTS. A grid was applied to the monitor providing 100 intersection points superimposed on the image of muscle cells and interstitial tissue. The number of nonmuscle areas (from a possible number of 100 intersections) was expressed as a percent of the blue-staining fibrotic area. The results of the fibrotic area were reported as means ± SD in square millimeters. Volume was calculated by multiplying the fibrotic area with the depth of penetration of fibrosis into the ventricular wall in serial sections of the heart from the epicardium to the endocardium. Further qualitative assessment of fibrosis was noted as either focal, diffuse, interstitial, or perivascular.
Aortic wall thickness was measured in three pairs of nontransgenic and transgenic mice. The ascending aorta used in analysis was embedded in paraffin and cut at 4-µm intervals until the aortic cusps were reached. Staining with HE and MTS was performed on two sections from the aortic root, two from the aorta proximal to the carotid bifurcation and two from the middle of the root and bifurcation of the ascending aorta. The slides of sections were imaged at x200 magnification, and aortic wall thickness was measured at five different points of the aortic circumference using Image-Pro Plus software. The means ± SD in micrometers were used in analysis.
Blood pressure measurements. Systolic blood pressure measurements were performed in conscious mice that had been acclimatized to a restrainer by the tail-cuff method (IITC Life Sciences Instruments, Woodland Hills, CA). Mice were placed in a temperature-controlled restrainer on a warm pad for 30 min before measurements were taken. A mean of a minimum of three readings was taken and graphed. Data were stored and analyzed using the ITTC computer software.
Echocardiography. Adult mice at 6 mo of age were anesthetized with intraperitoneal injection of ketamine (50 mg/kg) and xylazine (4 mg/kg). The ventral chest was shaved, and the mouse was placed on a thermally controlled foam pad. Echocardiography was performed using a Hewlett-Packard Sonos 5500 ultrasound imaging system equipped with a 10-MHz pulsed array transducer. Electrocardiogram leads (1 front paw and 2 hind paws) were placed. Conventional two-dimensional imaging, M-mode recordings, and spectral color Doppler evaluations were performed. Cardiac size and shape were determined using M-mode and two-dimensional image recordings. The LV wall thickness, contractility, and chamber dimensions were determined at end diastole and end systole. All values were based on the average of at least three consecutive beats to minimize noise and respiratory variation. Derivative measurements included LV mass, LV volume, and systolic function. Spectral Doppler recordings of mitral inflow patterns were used for evaluation of LV diastolic filling parameters.
Data analysis. Values were expressed as means ± SD. Data were analyzed by two independent observers blind to the transgenic status of the mice. Normality testing was performed on all data, and the t-test was used to determine the significance of differences between the two groups. When the data did not pass normality testing, the results were evaluated by the nonparametric Mann-Whitney U test and the equivalent Kruskall Wallis test for ANOVA. The criterion for significance was 0.05. Bonferonni correction was applied to multiple comparisons. A linear regression analysis of echocardiographic data from wild-type mice was performed with the E/A ratio as a dependent and age as an independent variable (Sigma Stat software). An R of >0.8 and a P value of 0.05 was considered significant in the regression analysis.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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After multiple microinjections were performed, the transgenic mice with
mild SRF overexpression and one single transgene copy number were obtained.
Northern blotting revealed that the levels of cardiac SRF mRNA were elevated
(Fig. 1). Quantitation of the
SRF mRNA level revealed that the transgenic mice had a mild (49%)
increase of cardiac SRF mRNA relative to age-matched nontransgenic animals.
These transgenic mice with 49% SRF overexpression had the lowest level of SRF
overexpression among all the SRF transgenic mouse lines that have been
produced to date in our laboratory
(56).
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Changes in gene expression in transgenic mice. Changes in cardiac
gene expression were observed in transgenic mice, which included a 50%
increase in skeletal -actin (SKA), a 400% increase in ANF, a 10%
increase in -MHC, and a 50% decrease in SERCA2 compared with the
nontransgenic mice (Fig.
1).
SRF expression in young and old nontransgenic mice. We also evaluated the expression of SRF mRNA in 3- and 21-mo-old wild-type mice and found that the 21-mo-old mice had 16% greater SRF mRNA expression compared with the 3-mo-old (Fig. 2, P < 0.05) mice.
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Cardiac morphological changes in transgenic mice. There were no obvious differences between the transgenic and nontransgenic mouse hearts at 6 mo of age. However, the heart weight-to-body weight ratio was slightly increased in transgenic (6.03 ± 1.0) compared with nontransgenic (5.03 ± 0.5, n = 8, P < 0.05) mice (Fig. 3). Measurement of ventricular wall thickness in the fixed sections using a micrometer under light microscopy revealed a slight increase of wall thickness in transgenic [17% increase in LV, n = 5, P < 0.05; 7.7% increase in septum, n = 5, not significant (NS); and 9.5% increase in RV, n = 5, NS, respectively] relative to nontransgenic mice (Fig. 4). The cardiac myocytes of the transgenic mice were heterogeneous in size, but most cells appeared to be larger in size than those of age-matched nontransgenic littermates (Fig. 5A). Measurement of the average cell size of cardiac myocytes, based on the cross-sectional diameter of the cardiomyocytes (Fig. 6), revealed a 12% increase in transgenic relative to nontransgenic mice (n = 1,000, P < 0.001).
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There was an increase in the collagen deposition of 6.7% (NS) observed in the hearts of the transgenic relative to the nontransgenic mice (Fig. 5B). The volume percentage of collagen increase in the transgenic mice was 6.7% more than that of the nontransgenic (NS). Collagen deposition was diffuse interstitial and present mainly in the epicardial region.
Aortic wall thickness as measured in age-matched (6 mo old) nontransgenic mice was 173 ± 35 µm (n = 3) and in transgenic mice was 168 ± 57 µm(n = 3). There was no significant difference between the two groups in the aortic wall thickness.
Cardiac morphological changes in 3-mo-old vs. 21-mo-old nontransgenic mice demonstrated a difference in morphology that was similar to that of the 6-mo-old nontransgenic compared with the age-matched transgenic mouse. Although the aging changes were more advanced in the hearts of 21-mo-old nontransgenic mice compared with those of the 6-mo-old transgenic mice, the trend was similar.
The heart weight-to-body weight ratio of the 3-mo-old nontransgenic was 3.7 ± 0.5 compared with 5.6 ± 0.8 in the 21-mo-old (n = 3, P < 0.05) mice. Measurement of LV wall thickness showed a 24% increase (n = 3, P < 0.05) in 21-mo-old relative to 3-mo-old mice. Measurement of the average cell size of cardiac myocytes, as based on the cross-sectional diameter of the cardiomyocytes, revealed an 18% increase in the 21-mo-old relative to that in 3-mo-old mice (n = 1,000, P < 0.001). The volume of fibrosis in the 21-mo-old mice was 14% greater than that in the 3-mo-old mice.
Functional assessment. Systolic blood pressure, as measured by the tail-cuff method in the conscious mice, did not demonstrate any significant difference, with the 6-mo-old nontransgenic mice having a blood pressure of 123 ± 12 mmHg and the age-matched transgenic mice having that of 126 ± 18 mmHg.
Detailed evaluation of in vivo cardiac structure and function was performed using echocardiographic imaging and Doppler flow assessment techniques. The chamber dimensions were measured, and the derived calculations of LV mass and volume as well as fractional shortening and relative wall thickness were made to gain a better characterization of the physiological consequences of mild cardiac specific overexpression of the SRF gene. LV diastolic filling parameters were also determined. As shown in Table 1, there was no difference between nontransgenic and transgenic F1 young adult animals at the age of 6 mo in terms of body weight or estimated LV mass.
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Evaluation of LV performance revealed that compared with age-matched nontransgenic mice, the young adult transgenic mice at 6 mo of age displayed characteristic cardiac functional changes resembling those that are usually observed much later in life in the aged human heart. The changes in the young adult transgenic mice included slightly increased diastolic posterior wall thickness, as well as significantly decreased LV diastolic and systolic dimensions (P < 0.01, and P < 0.01, respectively). Along with the changes observed above, the end-diastolic and end-systolic volumes were also decreased, whereas the relative wall thickness was increased in the young adult transgenic compared with nontransgenic littermates (P < 0.01, P < 0.01, and P < 0.01, respectively). Also, the parameters of pumping capacity such as stroke volume (ml/beat) decreased in transgenic compared with nontransgenic animals (P < 0.01). Significant differences were also observed between the transgenic and nontransgenic animals in the peak E wave and E/A ratio (P < 0.01 and P < 0.05, respectively).
These findings demonstrate that in young adult transgenic mice with mild SRF overexpression, there was evidence of mildly altered cardiac systolic and diastolic dimensions and function in terms of reduced LV stroke volume, as well as slightly delayed LV filling. However, there was no evidence of clinically significant cardiac hypertrophy, cardiac dysfunction, and/or congestive heart failure.
To confirm that the E/A ratio in the young adult transgenic mice was similar to that of older wild-type mice, we used the echocardiographic data from wild-type mice at different ages to perform a linear regression analysis of E/A ratio with age using Sigma Stat software. Our results demonstrated a significant association of E/A ratio with age, with an R of 0.869 and a P < 0.001 (Fig. 7). Interestingly, the hearts of 6-mo-old transgenic mice had an E/A ratio that matched that of older wild-type mice.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Aging is a complex biological process associated with a progressive decline in the physiological and biochemical performance of individual tissues and organs, leading to increased susceptibility to age-associated disease and functional senescence (26, 37, 54, 55). Multiple factors are likely involved in this life-long process. However, it is plausible that changes in a single gene level could potentially result in certain changes and/or syndromes that mimic aspects of accelerated aging, as has been observed with the mutations in the Werner's, Bloom's, and Ataxia-Telangiectasia genes (25, 30, 31, 33). Recently, a few mouse models with phenotypes that resemble various aspects of aging have been created. For example, defects in the klotho gene cause mice to die prematurely with a number of disorders commonly found in elderly people, such as arteriosclerosis, osteoporosis, skin atrophy, and emphysema (21). Targeted mutations in the first six exons of the p53 gene in the mouse can result in reduced life span, osteoporosis, generalized organ atrophy, and/or diminished stress tolerance. In addition, transgenic overexpression of a truncated form of the p53 gene results in an aging phenotype that is similar to that which has been observed in the p53 knockout mice (53). Delineation of the role of important transcription factors in the aging process using transgenic or gene-targeting approaches could significantly enrich our understanding of the process of normal adult aging.
The efforts of our laboratory have focused on understanding the mechanisms
of cardiac aging. The present study indicates that it is feasible to create a
transgenic mouse model with changes in cardiac function and morphology
resembling those of normal adult aging by mild cardiac-specific overexpression
of an important transcription factor such as SRF. We have shown that SRF
expression in mice increased by 16% from 3 to 21 mo of age, which
suggested to us that the level of overexpression of SRF in a transgenic mouse
model needed be relatively low to mimic the aging process. We therefore
generated the SRF transgenic mice with only one copy number of SRF transgene
that overexpressed SRF in the heart by 49% vs. the nontransgenic control
mice.
Aging of the heart is associated with a number of morphological and functional changes (2, 37, 38, 46). These age-related alterations show variability among rats, mice, and other species studied. In humans too, there is a range of cardiac changes that might occur with normal aging in the heart. In the absence of disease, aging may alter cardiac function during both systole and diastole, especially a reduction in the early LV diastolic filling (16, 37, 46). However, these changes are usually "mild" and without clinical significance in the majority of elderly who are free of cardiac diseases; therefore, they may be referred to as part of "normal cardiac aging" (17, 37, 46). Nevertheless, age-related changes in the heart in the absence of overt clinical disease predispose the heart to develop pathological changes. These changes also reduce the reserve capacity of the heart and make it more vulnerable to injury.
Most components of the cardiovascular system undergo some degree of change with aging, and various morphological alterations have been attributed to aging (22, 38). The myocardium in older individuals is characterized by a loss of myocytes with subsequent hypertrophy of the remaining viable myocytes. Ventricular mass is usually preserved or may be slightly increased. Those surviving myocytes may contain multiple nuclei and increased copies of chromosomes (polyploidy). As myocytes are lost and fibroblasts continue to proliferate and produce collagen, the physical properties of the aging heart become altered (16, 34, 37, 45). The histological changes observed in the hearts of the transgenic mice in the present study included cardiomyocyte hypertrophy and slight interstitial fi-brosis and LV wall thickness, which indicate that these transgenic mice had some morphological features resembling cardiac aging. The morphological changes seen in the transgenic mice in our studies were not as marked as those seen in the studies of Anversa and colleagues (Olivetti et al., Ref. 34), which showed a 60% increase in myocyte size and a 22% increase in collagen content in the old rat hearts. However, we believe that the results of Anversa and colleagues are representative of the extreme end of aging in rats, whereas ours represent an earlier phase of aging in otherwise healthy, young adult mouse hearts.
Interestingly, the echocardiographic changes observed in the young adult
transgenic mice with mild SRF overexpression at 6 mo of age mirrored those
that have been observed in older mice and also in elderly persons. For the
majority of older individuals who are free of clinically significant cardiac
disease, the aging heart adapts and performs its required functions fairly
well in the basal state (9,
23,
37). However, certain cardiac
changes have been observed in the healthy elderly, which include increased LV
end-diastolic relative wall thickness, decreased early diastolic filling,
increased duration of myocardial relaxation, increased myocardial stiffness,
decreased responsiveness to -adrenergic agonists, decreased arterial
compliance, decreased maximum aerobic capacity, and decreased baroreceptor
reflex sensitivity (37,
39).
The prolonged contraction duration and myocardial relaxation phase observed in the heart of the older mammal is partly due to prolonged calcium entry during an extended sarcolemmal depolarization, as well as decreased velocity of calcium uptake from the sarcoplasmic reticulum after depolarization. Age-related decreases in the activity of the sarcoplasmic reticulum pump (ATPase) are correlated with age-associated changes in SERCA2 gene expression (4, 27, 37). The SRE is in the promoter region of SERCA2. SRF binds to SRE and regulates the expression of SERCA2 gene (6). In the present study, we demonstrated that mild overexpression of SRF resulted in downregulation of cardiac SERCA2 mRNA in the transgenic mice. This finding supports the notion that the mildly increased SRF that has been observed in the heart of the older animal (28, 52) might contribute to the age-associated decrease of SERCA2 level in senescence and thereby also contribute to the age-associated prolongation of cardiac relaxation.
The increase in the expression level of ANF and skeletal actin in our
transgenic mouse model in this project parallels the increase seen in these
genes in other cardiac hypertrophic conditions in which reexpression of the
fetal gene program occurs
(22). The increase in
-MHC observed in our transgenic mouse is also similar to that observed
during normal cardiac aging
(37).
The combination of prolonged cardiac relaxation and increased myocardial stiffness during adult aging may result in an elevated LV end-diastolic pressure at rest and with exertion (1, 5). This has also been associated with the characteristic finding of decreased early diastolic filling in elderly individuals. Because early diastolic filling is reduced, there is consequently relatively more filling during late diastole in old compared with younger individuals. These changes are demonstrated on Doppler echocardiography as a change in the ratio of E/A ratio (7, 37). A long-term follow-up study has reported that E/A ratio changes during aging and drops more than one-half from the 30s to the 80s during a person's lifetime (9). It has been considered that the E/A ratio is a sensitive and important indicator of cardiac aging (8-10, 15-17, 20, 29, 35, 36, 38, 40, 46, 48, 49). In the current study, the young adult transgenic mice had a 20% decline in peak E and a 35% decrease in the E/A ratio relative to the nontransgenic mice. Our result of an E/A ratio of 1.84 ± 0.31 is larger than the E/A ratio of 1.55 ± 0.07 reported by Taffet et al. (46). However, the mice used in the study by Taffet et al. were significantly older (32 mo), and it is likely that transgenic mice at a slightly older age than 6 mo (perhaps 9-11 mo) would have had a lower E/A ratio. A linear regression analysis of E/A ratios of other healthy wild-type mice of different ages performed in our laboratory demonstrates a significant correlation between E/A ratio and age, with an R of 0.869 and a P < 0.001 (Fig. 7). In addition, the E/A values of the older wild-type mice in the current study (age 36 mo) are in agreement with those reported by other observers (10, 16, 20, 40, 46, 48).
In summary, aging is a process that spans many decades in human beings and, similarly, many months in rodents. To translate human cardiac aging with a much longer life span into rodent years may be fraught with complexities. However, in general, aging might be arbitrarily divided into an earlier phase (humans between 50 and 60 years and in rodents between 10 and 17 mo), a middle phase (humans between 61 and 80 years and in rodents between 18 and 24 mo), and a late phase (humans between 81 and 100 years and in rodents between 25 and 32 mo). Of course, with the progressively changing demographics and aging of the population, these arbitrary divisions of what might be considered "early" or "late" aging will likely undergo revision and change. Currently, the most consistently characteristic change studied in individuals in the mid-phase of aging is the reduced cardiac E/A ratio. Our transgenic mice at a chronological age of 6 mo demonstrate this E/A reduction. Hence, the findings from the present study suggest that the age-associated increase in SRF expression that has been observed in rodents has functional significance and likely contributes to the changes considered to be characteristic of the aging heart. The cardiac-specific overexpression of SRF at a low level has resulted in the creation of a mouse model of myocardial aging, with an "old heart" in a young adult body. This model could potentially help to further elucidate the molecular mechanisms of human cardiac aging.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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-MHC promoter, Dr. R. Prywes
for pCGNSRF plasmid, and Dr. J. Lawitts for DNA microinjection. We thank K.
Conover for echocardiographic analysis and S. White for histological
analysis. |
FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* X. Zhang and G. Azhar contributed equally to this work. 
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) had an E/A ratio resembling that of the old mice. NTg littermate
(
) mice had an E/A ratio that was normal for their age as depicted in the
linear regression. E/A ratios from all mice except the SRF Tg were used in the
regression analysis. R = 0.869, r2= 0.755, and P
< 0.001