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Structural and functional alterations in the senescent heart have been associated with an activated sympathetic nervous system and a regional cardiac renin-angiotensin system. To date, however, limited information related to their expression alteration during the whole pro-cress of growth and development has been reported.
To examine the expression of alpha(1)-adrenergic receptor (α1-AR) and angiotensin II receptor (ATR) subtypes in the left ventricle of hearts from young adult, middle-aged, presenescent and senescent rats.
Semiquantitative reverse transcriptase polymerase chain reaction and Western blot were used to quantitate the messenger RNA and protein of α1-AR and ATR subtypes, respectively, in the left ventricles of three- (young adult), 12- (middle age), 18- (presenescent) and 24-month-old (senescent) Wistar rats.
α1A-AR expression decreased gradually with age, and α1D-AR expression was repressed in middle age and presenescence, while the expression of α1B-AR remained unchanged during senescence. AT1R expression was unaffected by aging from young adulthood to presenescence, but exhibited a remarkable upregulation in senescence. There were no significant discrepancies of cardiac AT2R expression among the four age groups, but both messenger RNA and protein had a tendency to upregulate during aging. The results suggest that there are considerable changes of expression of cardiac α1-AR and ATR subtypes during growth and development. The change of cardiac α1-AR and ATR expression during aging is a protective response to senescence by keeping normal myocardial contractility, while the upregulation of AT1R and AT2R promotes age-related myocardium hypertrophy and cardiac remodelling.
Les altérations structurales et fonctionnelles du cœur scénescent ont été associées à un système nerveux sympathique activé et à un système rénine-angiotensine cardiaque régional. À ce jour, toutefois, peu de données ont mentionné une altération de leur expression durant le processus complet de la croissance et du développement.
Examiner l’expression des sous-types de récepteurs alpha1-adrénergiques (RA-α1) et de récepteurs de l’angiotensine II (AT) dans le ventricule gauche de cœurs de rats adultes jeunes, d’âge moyen, présénescents et sénescents.
La réaction en chaîne de la polymérase semi-quantitative par transcriptase inverse et le transfert Western ont été utilisés respectivement pour quantifier l’ARN messager et les protéines des sous-types RA-α1 et AT dans les ventricules gauches de rats Wistar de trois mois (jeunes adultes), 12 mois (âge moyen), 18 mois (présénescents) et 24 mois (sénescents).
L’expression des RA-α1 a graduellement diminué avec l’âge et l’expression des RA-α1D a été réprimée à l’âge moyen et en présénescence, tandis que l’expression des RA-α1B est restée inchangée durant la sénescence. L’expression des récepteurs AT1 n’a subi aucune influence du vieillissement entre l’état de jeune adulte et la présénescence, mais a manifesté une remarquable régulation à la hausse à la sénescence. On n’a noté aucun écart significatif quant à l’expression des récepteurs AT2 cardiaques entre les quatre catégories d’âge, mais l’ARN messager et les protéines avaient tendance à être ajustés à la hausse avec le vieillissement. Ces résultats suggèrent l’existence de changements considérables de l’expression des sous-types de RA-α1 et de récepteurs AT cardiaques durant la croissance et le développement. Les variations de l’expression des RA-α1 et des récepteurs AT cardiaques durant le vieillissement sont une réponse protectrice contre la sénescence, qui vise à maintenir la contractilité myocardique normale, tandis que la régulation à la hausse des récepteurs AT1 et AT2 favorise l’hypertrophie myocardique liée à l’âge et le remodelage cardiaque.
Several receptor systems coexist in the heart, including the adrenergic receptor (AR) and the angiotensin II receptor (ATR). Among the cardiac adrenoceptor subtypes, it is well known that beta (β)-AR is predominant with respect to mediating positive inotropic and chronotropic effects (it accounts for approximately 75% of the inotropic response); however, a vast body of evidence has accumulated showing that the alpha(1)-AR (α1-AR) also plays an important role in causing positive inotropic effects (it accounts for approximately 25% of the inotropic response). A recent finding (1) indicates that, in addition to the regulation of myocardial contractility, α1-AR is involved in mediating the hypertrophic response of cardiomyocytes. Moreover, it has been shown that the ATR not only influences hemodynamics, but also regulates the growth and hypertrophic response of cardiomyocytes, vascular smooth muscle cells and fibroblasts, which are related to cardiovascular remodelling and functional alteration.
The endogenetic ligands of α1-AR are noradrenaline and adrenaline, while the ATR is always activated by angiotensin II (Ang II). According to its pharmacological characteristics, α1-AR is divided into three subtypes: α1A-AR, α1B-AR and α1D-AR. ATRs can be grouped into AT1R and AT2R. Structural and functional alterations in the senile heart have been associated with an activated sympathetic system and a regional cardiac renin-angiotensin system. However, the influence of aging on the expression of cardiac α1-AR and ATR subtypes has remained uncertain. Previous investigations (2–4) focused on comparing the expression of α1-AR and ATR subtypes between young adulthood and senescence, but to date, limited information has been available with respect to their expressive alteration during the process of growth and development. Thus, we examined the expression of α1-AR and ATR subtypes in the left ventricle of young adult, middle age, presenescent and senescent rats.
Twenty-four male Wistar rats (Vital River Lab Animal Technology Co Ltd, China), fed in the animal room of the Beijing Anzhen Hospital (Beijing, China), were used for the experiments. The rats were divided into four groups based on age: three months of age (n=6; weight 321 g to 375 g), 12 months of age (n=6; weight 690 g to 793 g), 18 months of age (n=6; weight 831 g to 974 g) and 24 months of age (n=6; weight 918 g to 1029 g). These groups represented young adulthood, middle age, presenescence and senescence, respectively. After anesthesia via an abdominal injection of 1.5% pentobarbital (45 mg/kg), the hearts were removed and the left ventricles were separated; then, all samples were stored at −80°C until use. Goat antirat α1A-AR, α1B-AR and α1D-AR antibodies were purchased from Santa Cruz Biotechnology Inc (USA). Rabbit antirat AT1 and AT2 antibodies were purchased from United States Biological (USA). TRIzol kits were obtained from Gibco Corporation (USA). Moloney murine leukemia virus reverse transcriptase (RT), deoxyribonucleoside triphosphates and polymerase chain reaction (PCR) reagents were obtained from Promega Corporation (USA). Sense and antisense oligonucleotide primers for RT-PCR were synthesized by Bejing Sunbiotech Co Ltd (China).
Total RNA was extracted from the left ventricles using the TRIzol kit, and an ultraviolet spectrophotometer was used to measure the concentration of total RNA in each sample. Complementary DNA was synthesized and amplified using a PCR kit (PE2400, Applied Biosystems, USA). To quantify the transcripts by RT-PCR amplification, glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal standard and the target messenger RNA (mRNA) was normalized with GAPDH. The primer pair sequences for GAPDH were 5′-TGCACCACCAACTGCTTAGC-3′ (sense) and 5′-GGCATGGACTGTGGTCATGAG-3′ (antisense). The PCR reaction parameters were an initial denaturing at 94°C for 45 s, annealing at 55°C for 45 s, then extension at 72°C for 45 s (25 cycles). The primer pair sequences for α1A-AR were 5′-CAAGGCCTCAAGTCCGGCCT-3′ (sense) and 5′-CTCTCGAGAAAACTTGAGCAG-3′ (antisense). The reaction parameters were an initial denaturing at 94°C for 45 s, annealing at 57°C for 45 s, then extension at 72°C for 45 s (35 cycles). The primer pair sequences for α1B-AR were 5′-ATCGTGGCCAAGAGGACCAC-3′ (sense) and 5′-CTCTCGAGAAAACTTGAGCAG-3′ (antisense). The reaction parameters were an initial denaturing at 94°C for 45 s, annealing at 58°C for 45 s, then extension at 72°C for 45 s (35 cycles). The primer pair sequences for α1D-AR were 5′-CGTGTGCTCCTTCTACCTACC-3′ (sense) and 5′-GCACAGGACGAAGACACCCAC-3′ (antisense).The reaction parameters were an initial denaturing at 94°C for 45 s, annealing at 63°C for 45 s, then extension at 72°C for 45 s (28 cycles). The primer pair sequences for AT1R were 5′-CGTCATCCATGACTGTAAAATTTC-3′ (sense) and 5′-GGGCATTACATTGCCAGTGTG-3′ (antisense). The reaction parameters were an initial denaturing at 94°C for 45 s, annealing at 61°C for 45 s, then extension at 72°C for 45 s (30 cycles). The primer pair sequences for AT2R were 5′-GTGTGGGCCTCCAAACCATTGCTA-3′ (sense) and 5′-TTGCTGCCACCAGCAGAAAG-3′ (antisense). The reaction parameters were an initial denaturing at 94°C for 45 s, annealing at 53°C for 45 s, then extension at 72°C for 45 s (33 cycles). The amplified RT-PCR products were α1A-AR (156 base pairs [bp]), α1B-AR (287 bp), α1D-AR (304 bp), AT1R (236 bp) and AT2R (179 bp). 10 μL PCR products were electrophoresed on 1.5% agarose gel, and then viewed by an Alpha imager 2200 (NatureGene Corporation, USA). Densitometry was used in the relative semiquantitative assessment of RT-PCR products.
After homogenizing heart tissue, total protein was extracted. A coomassie brilliant blue assay was used to determine the concentration of total protein. The proteins were resolved by 10% sodium dodecyl sulfate polyacrylamide gel, transferred to a nitrocellulose filter membrane using a semidry electrophoretic graphite electrode with a constant current of 1 mA/cm2 gel for 1 h, and probed with the diluted specific antibody (the diluted concentrations of specific antibodies for receptor subtypes were as follows: α1A-AR, 1:1000; α1B-AR, 1:1000; α1D-AR, 1:500; AT1R, 1:10000; AT2R, 1:7500), followed by horseradish peroxidase conjugated secondary antibody (β-actin was used as an internal standard). An antibody chromogenic agent (Santa Cruz Biotechnology Inc, USA) was placed on the membrane for the development of a film and a scanner was used for imaging. Densitometry was used to measure the protein level.
Densitometry for RT-PCR and Western blot images was performed using BandScan software (Glyko, USA). Measured mRNA or protein levels were normalized to the internal standard (GAPDH or β-actin) and expressed as a ratio of the particular receptor to the internal standard. Data are presented as means ± SDs in all assays. SPSS 12.0 (SPSS Inc, USA) was used for the statistical analysis. Statistical significance was estimated by one-way ANOVA followed by the Newman-Keuls test for group-to-group comparisons. The test was considered significant at P<0.05.
Body weight (BW) and heart weight increased remarkably during aging, while left ventricle weight was augmented in middle age versus in young adulthood, and remained stable in presenescence and senescence. For the marked increase of BW during aging, the heart weight to BW and left ventricle weight to BW ratios were diminished in middle age, presenescence and senescence versus young adulthood, whereas there were no significant changes observed among the latter three age groups (Table 1).
RT-PCR analysis showed the expression of α1A-AR in different age groups (Figure 1 and Table 2). There was remarkable downregulation in the 12-, 18- and 24-month-old groups compared with the three-month-old group (P<0.01). Moreover, the mRNA level was significantly decreased in the 24-month-old group compared with the 18-month-old group (P<0.01), while it remained unchanged between the 12- and 18-month-old groups (P>0.05).
RT-PCR analysis showed the expression of α1D-AR in the different age groups (Figure 3 and Table 2). The mRNA level was decreased in the 12-month-old group (P<0.05), and was drastically decreased in the 18- and 24-month-old groups compared with the three-month-old group (P<0.01). Furthermore, in the 18-month-old group, the mRNA level also decreased markedly relative to the 12-month-old group (P<0.01), while there was no significant change between the 18- and 24-month-old groups.
Western blot analysis showed the expression of α1A-AR in the different age groups (Figure 4 and Table 3). There was remarkable down-regulation in the 12-, 18- and 24-month-old groups compared with the three-month-old group (P<0.01). A significant decline of α1A-AR expression was also observed in the 18-month-old group compared with the 12-month-old group (P<0.01) and in the 24-month-old group compared with the 18-month-old group (P<0.01).
Western blot analysis showed the expression of α1D-AR in the different age groups (Figure 6 and Table 3). There was considerable downregulation in the 12-, 18- and 24-month-old groups compared with the three-month-old group (P<0.01). Protein level was also markedly decreased in the 18-month-old group compared with the 12-month-old group (P<0.01), while no significant difference was observed between the 18- and 24-month-old groups (P>0.05).
RT-PCR analysis showed the expression of AT1R in the different age groups (Figure 7 and Table 2). There was notable upregulation in the 24-month-old group relative to the three-, 12- or 18-month-old groups (P<0.01), but no significant difference was observed among the three younger age groups (P>0.05).
Western blot analysis showed the expression of AT1R in the different age groups (Figure 9 and Table 3). AT1R protein level increased in the 24-month-old group relative to the three-, 12- and 18-month-old groups (P<0.05), but no significant difference was observed among the three younger age groups (P>0.05).
As shown in Table 4, a significant linear correlation between mRNA levels and protein levels of α1-AR and ATR subtypes was observed.
Previous studies have shown that α1-AR and ATR subtypes coexist in the heart (5). α1-AR has an essential role in the heart via various mechanisms, including positive inotropic and chronotropic effects, physiological growth or pathological hypertrophy of cardiomyocytes, apoptosis and cardiac remodelling. α1A-AR is principally responsible for facilitating hypertrophy of cardiomyocytes (1) and myocardial contractility (6). α1D-AR, which is mainly distributed in the vascular system, has an effect on regulation of coronary flow (7). α1B-AR is associated with mediating the inotropic response (6). AT1R stimulation induced by Ang II influences not only hemodynamics but also promotes hypertrophy of cardiomyocytes, proliferation of fibroblasts and cardiac interstitial fibrosis. AT2R has a regulatory role in cardiomyocyte growth and hypertrophy (8,9).
The present study investigated the alteration of mRNA and protein levels of α1-AR subtypes in the whole process of growth and development. The results demonstrated that α1A-AR expression was downregulated gradually with aging and α1D-AR expression was decreased in middle age and presenescence, while α1B-AR expression remained unchanged. These findings were consistent with results of studies by Yu et al (2) and Zhang et al (10). As shown in Table 4, during senescence, there was a remarkable linear correlation between mRNA levels and protein levels of α1-AR subtypes, indicating that decreased gene transcriptional activity may be the major reason for downregulation of receptor proteins.
The first question involved the possible influence of the change of α1A-AR and α1B-AR expression during aging on the growth response of cardiomyocytes. First, a series of studies (1,11–13) showed that α1A-AR and α1B-AR might be the receptor subtypes for noradrenaline-induced cardiomyocyte hypertrophy, while it remained uncertain which of these two subtypes predominated, whether either of them was indispensable, and whether one played a complementary role to the other. Second, cardiac hypertrophy was assumed to be an age-associated change, both in human and animal models. In elderly persons who were apparently healthy, left ventricular hypertrophy seemed to be a trigger for impaired diastolic function, changed diastolic filling pattern, increased end diastolic volume and even terminal heart failure. Third, we observed a considerable downregulation of α1A-AR with aging. Thus, we propose that the decline of cardiac α1A-AR expression associated with aging might be an adaptive response to senescence to attenuate the effects of α1A-AR and repress catecholamine-induced myocardial hypertrophy. The finding that the protein level of α1A-AR was downregulated in presenescence compared with in middle age was not consistent with the unchanged mRNA level observed in this age group. This finding might result from slower progression from mRNA into protein or accelerated mRNA degradation.
The second problem related to the possible effects of the down-regulated α1A-AR and unaffected α1B-AR expression level during senescence on myocardial contractility. We hypothesize that the downregulated α1A-AR and unchanged α1B-AR expression helps maintain normal contractile function in the aged heart via interaction between α1-AR and β-AR. The myocardial inotropic effect was mediated jointly by α1-AR and β-AR, and respective activation of α1-AR or β-AR could produce a positive inotropic effect, although β-AR might be predominant. Moreover, a physiological interaction between α1-AR and β-AR has been discovered. Subtype-specific interaction has also been characterized such that α1A-AR could repress the positive inotropic effect mediated by β-AR, while α1B-AR could potentiate. Therefore, downregulation of α1A-AR expression during aging may diminish the restraint to β-AR, and maintenance of α1B-AR expression level might, to some extent, help maintain normal myocardial contractility.
The third aspect dealt with α1D-AR. It was recently shown that α1D-AR stimulation has no direct influence on myocardial inotropic and chronotropic effects (14), but can impair myocardial contractility by reducing coronary flow (7). The downregulation of cardiac α1D-AR expression in middle age and presenescence demonstrated in our experiments might be an adaptive response to senescence, which could contribute to maintain normal coronary flow by means of decreasing α1D-AR gene transcription activity and receptor content, to prevent the occurrence of myocardial ischemia.
The mechanism of α1A-AR and α1D-AR expression downregulation during senescence remains unclear. Nevertheless, we believe the reason for decreased α1A-AR and α1D-AR expression might be that the sympathetic nervous system activity was elevated in senescence, and a senescence-related mechanism increased regional cardiac concentrations of catecholamines via an autocrine or paracrine process. Alternatively, expression of α1-AR could be influenced by an interaction of cardiac receptors. On the one hand, it was observed that AT1R stimulation induced by Ang II could decrease α1A-AR mRNA levels and protein density in cardiomyocytes (15). On the other hand, there were elevated levels of angiotensinogen and angiotensin-converting enzyme mRNAs in the senescent left ventricle, despite a lower circulating level (16). Thus, activation of the renin-angiotensin system in the aged heart may restrain α1A-AR expression via AT1R. In short, alteration of cardiac α1-AR gene expression was synthetically influenced by the factors mentioned above. Elucidating its intrinsic mechanism requires further research.
As shown in Table 4, similar to α1-AR, a significant linear correlation between mRNA and protein levels of ATR has been discovered. It also suggests that the change in expression of these receptors is due to transcriptional regulation. In the present study, cardiac AT1R expression did not significantly change from young adulthood to presenescence, but exhibited a remarkable upregulation in senescence. This result was consistent with those reported by Heymes et al (3). Primarily, the Ang II-induced hypertrophic effect via AT1R was another pathway for mediating myocardial hypertrophy and even age-associated myocardial hypertrophy, and it has been reported that AT1R blockade decreased age-associated left ventricular hypertrophy, independent of blood pressure-lowering effects (8). The considerable upregulation of cardiac AT1R in senescence could, at least partially, account for age-associated cardiac hypertrophy. Another effect of the rise of AT1R expression on the heart might be compensating for the decreased contractility in the senescent heart via interaction between cardiac ATR and α1-AR in mediating a positive inotropic effect. A recent study (17) demonstrated that there was an age-related difference in the interaction. Ang II had no positive cardiac inotropic effect induced by phenylephrine (PE) in young adult and middle-aged subjects, whereas it could promote a PE-induced positive inotropic effect in presenescence and senescence. As the principal functional receptor subtype mediated by Ang II, the considerable upregulation of AT1R, accompanied by an elevated cardiac Ang II level (16) in senescence may potentiate α1-AR effects and, to some extent, counteract the negative effects with respect to myocardial contractility brought on by the downregulation and desensitization of α-AR and remodelling of the cardiovascular system in senescence. However, it is difficult to explain the potentiated PE-induced positive inotropic effect in presenescence (as repored by Shi et al ) by the unchanged AT1R expression, as the present study showed in this age group. We believe that such a discrepancy between function and expression might be due to different ATR expression levels between the left ventricle and atrium (17).
The results of our experiments show there were no significant differences in cardiac AT2R expression among the four age groups. However, both mRNA and protein levels had a tendency for upregulation during aging (Tables 1 and and2).2). The reason that the results revealed no change among the different age groups might be the small number of samples, to some extent. If we had included more samples in the study, positive results might have been elicited. The tendency for upregulation of cardiac AT2R expression during senescence was consistent with the results of Heymes et al (3), and the consistency between mRNA and protein levels of AT2R could be the most persuasive support for our conclusions. AT2R-mediated physiological effects were considered to be antagonizing that of AT1R, but this concept was recently questioned. It was reported that AT2R had a capacity for promoting cardiomyocyte hypertrophy and had no direct antagonistic action on AT1R in mediating cardiomyocyte growth (9). Thus, as effects of the rise of cardiac AT1R expression, the upregulation of AT2R in the senescent heart and the elevated cardiac Ang II level (16) might contribute to age-related myocardial hypertrophy and cardiac remodelling.
Taken together, the expression of both cardiac α1-AR and ATR subtypes was altered in the direction that maintained normal myocardial contractility, whether through the downregulation of α1A-AR and α1D-AR, no change in α1B-AR or the upregulation of AT1R. Moreover, the decline of α1A-AR could reduce catecholamine-induced myocardial hypertrophy, while the rise of AT1R and AT2R expression might contribute to cardiomyocyte hypertrophy. Therefore, the changes of cardiac α1-AR and ATR expression during aging may be a protective response to senescence with respect to maintaining normal myocardial contractility. However, with respect to mediating cardiomyocyte hypertrophy, the meaning of the foresaid changes still remains uncertain.
α1-AR and ATR subtypes coexist in the heart, and each of them plays an important role in regulating cardiac structure and function. With aging, the altered expression levels of these two groups of receptors has been observed, but more details about the meaning and potential mechanism of the change of α1-AR and ATR expression during senescence will require further investigation.
SUPPORT: The present study was supported by the Beijing Municipal Natural Science Foundation (#7042056).