Expressions of cardiac sympathetic norepinephrine transporter and β₁-adrenergic receptor decreased in aged rats

Expressions of cardiac sympathetic norepinephrine transporter and β 1 -adrenergic receptor decreased in aged rats 1 2 first-author †E-mail:hli_tianjin@yahoo.com.cn †E-mail:hli_tianjin@yahoo.com.cn hli_tianjin@yahoo.com.cn 1 1 1 1 1 1 2 Cardiovascular Division, Department of Geriatric Medicine, Pingjin Hospital, Medical College of Chinese People’s Armed Police Force, Institute of Cardiovascular Disease of Chinese People’s Armed Police Force, Tianjin 300162, China Cardiovascular Division, Department of Geriatric Medicine, Pingjin Hospital, Medical College of Chinese People’s Armed Police Force, Institute of Cardiovascular Disease of Chinese People’s Armed Police Force, Tianjin 300162, China Cardiovascular Division, Changzheng Hospital, Shanghai 200003, China Cardiovascular Division, Changzheng Hospital, Shanghai 200003, China Evidence suggests that the deterioration of communication between the sympathetic nervous system and cardiovascular system always accompanies the aging of human and animals. Cardiac sympathetic norepinephrine (NE) transporter (NET) on presynaptic membrane is a predominant component to eliminate released NE in the synaptic cleft and maintains the sensitivity of the β-adrenergic receptor (β-AR). In the present study, we investigated NET and β 1 -AR mRNA levels and sympathetic nerve density in cardiac sympathetic ganglion and left ventricular myocardium in 2- and 16-month-old rats with Northern blot analysis and immunohistochemistry. The expression levels of NET mRNA, NET protein and β 1 -AR mRNA in the ganglia or myocardia of 16-month-old rats were markedly reduced by 67%, 26%, and 43%, respectively, in comparison with those in 2-month-old rats. Our results also show that aging induces a strong decrease of the catecholaminergic nerve fiber density. Norepinephrine transporter (NET) β 1 -adrenergic receptor (β 1 -AR) Cardiac sympathetic ganglion Aging INTRODUCTION 1 There are several lines of evidence suggesting that advanced aging in human and animals is accompanied by deterioration of communication between the sympathetic nervous system and cardiovascular system (Lakatta, 1993 1993 ; Esler et al., 1995 1995 ; Seals and Esler, 2000 2000 ; Li et al., 2003 2003 ). The efficacy of β-adrenergic modulation of cardiovascular function is decreased with aging, particularly during stress, and it has been thought that the age-associated deficit in the effectiveness of β-adrenergic control is largely postsynaptic, and that desensitization of β-adrenergic receptor (β-AR) responses occurs via modifications of both receptor and postreceptor events (Chevalier et al., 1991 1991 ; Sakai et al., 1992 1992 ; Lakatta, 1993 1993 ; White et al., 1994 1994 ; Esler et al., 1995 1995 ; Ferrara et al., 1997 1997 ; Hardouin et al., 1997 1997 ). However, it was reported that neither G-protein-coupled-receptor kinases (GRKs) nor inhibitory G-proteins (Gi proteins) appear to contribute to the age-associated reduction in cardiac β-AR responsiveness (Xiao et al., 1998 1998 ). The mechanism underlying this age-dependent reduction in cardiac β-adrenoceptor function is not completely understood. The norepinephrine transporter (NET) at presynaptic nerve terminals mediates the uptake of released norepinephrine (NE), resulting in the rapid termination of synaptic transmission and thereby controlling the fine tuning of neuronal activities (Esler et al., 1990 1990 ; Povlock and Amara, 1997 1997 ). Age-related reduction in reuptake of NE and β-AR density in the heart of health senescent human and animals was reported (Esler et al., 1990 1990 ; 1995 1995 ; Chevalier et al., 1991 1991 ; Dawson and Meldrum, 1992 1992 ; Sakai et al., 1992 1992 ; White et al., 1994 1994 ; Ferrara et al., 1997 1997 ; Hardouin et al., 1997 1997 ; Guimaraes et al., 1998 1998 ; Snyder et al., 1998 1998 ; Xiao et al., 1998 1998 ). Failure of transmitter inactivation at postjunctional receptors with aging would amplify the neural signal. Because the myocardial interstitial NE content depends predominately upon the amount released and reuptake capacity by the neuron, a defective NE uptake could contribute to high concentration in the synaptic cleft and β-AR density downregulation, even in situations in which the release of NE from the sympathetic cardiac neurons is low (Esler et al., 1990 1990 ; Povlock and Amara, 1997 1997 ). Thus, it is speculated reasonably that the reuptake capacity of NET might have declined with age and contributed to β-AR downregulation and age-related decreases of cardiac β-adrenergic responsiveness. The results of radioactive scanning for cardiac sympathetic neuroimaging have generally agreed with the notion of an age-related decline in cardiac uptake activity (Leineweber et al., 2002 2002 ; Kiyono et al., 2002 2002 ; Kaye and Esler, 2005 2005 ). However, there is little information regarding the speculation that changes of the upstream modulator of β-adrenergic responsiveness are involved in the age-related β-AR downregulation. Whether there are declined levels of both mRNA and protein of cardiac sympathetic NET with aging is not known at present. The aim of this investigation is to examine whether there is an age-related reduction in the cardiac NET and β 1 -AR mRNA expressions. MATERIALS AND METHODS 1 In this study, 2- ( n =10) and 16-month-old ( n =10) male Sprague-Dawley (SD) rats were used. Animal use procedures were followed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, USA. After acclimatization period, rats were sacrificed by decapitation, and the middle and inferior (stellate) sympathetic ganglia (middle cervical-stellate ganglion (MC-SG) complex) of both sides were isolated. The sympathetic ganglionic innervation of rat heart originates primarily MC-SG complex (Pardini et al., 1989 1989 ; 1990 1990 ), and expression of cardiac NET mRNA is in the ganglia (Li et al., 2001 2001 ). The hearts ( n =5, each age group) were dissected into the left and right ventricles, and the left ventricle was separated into two equivalent sections along its long-axis. The upper sections of left ventricular myocardium were quickly frozen in liquid nitrogen until the mRNA analysis performed. The lower sections of left ventricular myocardium were immersed and fixed with 0.04 g/ml paraformaldehyde at 4 °C, and the sections were cut into 18-µm thick slices for immunohistochemical staining for NET and adrenergic innervation of the heart. The total RNA was extracted from the MC-SG complex and LV myocardium using the reagent of TRIzol (Gibco BRL, Gaithersburg, MD, USA) according to the instructions. DNase I was used to eliminate contamination of genomic DNA in RNA preparation, and 1 µg of total RNA of the ganglia was used for reverse transcription (RT) into cDNA with Moloney murine leukemia virus reverse transcriptase (M-MLV) (Gibco BRL) according to standard methods (Li et al., 2001 2001 ). NET and β 1 -AR Northern blottings were performed as described previously (Li et al., 2001 2001 ). Briefly, 15 µg of total RNA of the ganglia and left ventricular myocardia of 2- and 16-month-old rats were electrophresed on a 0.01 g/ml agarose-formaldehyde gel and transferred to a Hybond-N membrane (Amersham, International, Buckinghamshire, UK). The blots were prehybridised for 3 h and hybridised for 20 h with 32 P-labeled NET or β 1 -AR cDNA as probe in 0.5 g/ml formamide at 42 °C. After autoradiography exposure, the blots were stripped and rehybridised with 32 P-labeled β-actin cDNA probe. Each hybridisation signal of specific band of NET and β 1 -AR was measured by densitometry, and was normalized by the corresponding hybridisation signals obtained from the β-actin cDNA probe to correct differences in loading and transfer. The β-actin signal was used as a correction factor. Probes were labeled by random priming with DNA labeling system (Boehringer Mannheim GmbH, Germany) and radioactivity of the probes at 1.8×10 9 cpm/µg. The NET probe is a 407-bp amplification product of the cloned rat NET gene (Kitayama et al., 1999 1999 ) between a sense primer 5′-CTC AAG GAG GCC ACG GTA TGG ATC G-3′ and an antisense primer 5′-ACC TGG AAG TCA TCA GCC AGT CCG G-3′, and β-actin probe is a 285-bp product of the murine β-actin gene between a sense primer 5′-TCA TGA AGT GTG ACG TTG ACA TCC GTA AAG-3′ and an antisense primer 5′-CCT AGA AGC ATT TGC GGT GCA CGA TGG AGG-3′ (Li et al., 2001 2001 ). β 1 -AR probe, a 900-bp Pst I coding fragment of the β 1 -AR genomic clone, was a kind gift of Dr. CA Machida (Division of Neurosicence, Oregon Regional Primate Research Center, Oregon Health Sciences University, USA). An avidin/biotin blocking kit (HISTOSTAIN™-DS Kit; Zymed Laboratories, San Francisco, CA, USA) was used for detection of NET expression. The sections of left ventricle were incubated in primary antisera:rabbit anti-NET (1:250 (v/v); Alpha Diagnostic Intl. Inc., San Antonio, USA) according to the manufacturer’s instructions (Zymed Laboratories). The immunohistochemical methods for tyrosine hydroxylase (TH) were used as previously described (Himura et al., 1993 1993 ) with modification. Briefly, the specimens were soaked in methanol with H 2 O 2 for 30 min. After washing in phosphate-buffered saline (PBS, 0.01 mol/L, pH 7.2), the specimens were reacted with normal goat serum (1:20; Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Then, they were incubated with anti-TH antibody (1:100; Sigma Chemical, St. Louis, MO, USA) for 24 h at 4 °C. After rinsing with PBS containing 0.5 mg/ml Tween 20, they were incubated with secondary antibody (goat anti-mouse IgG, 1:100; Vector Laboratories) conjugated to horseradish peroxidase for 1 h. After rinsing with PBS containing 0.5 mg/ml Tween 20, they were reacted with avidin-biotin complex (ABC) reagent (Vector Laboratories). Finally, they were reacted for 5~10 min with 0.5 mg/ml 3,3′-diaminobenzidine (Sigma Chemical) and 0.01% (v/v) H 2 O 2 in Tris buffer (pH 7.6). The sections were finally mounted on gelatin-coated slides, dried, dehydrated through a series of ethanol, cleared in xylene, and coverslipped in Permount. These specimens were microscopically examined, and the distribution densities of the NET and TH of LV myocardium were calculated by the point-counting method (Kawano et al., 2003 2003 ). Briefly, for each piece of myocardium, 10 fields were randomly selected in each area of a specimen, and a section was registered if the section contained the nerve fibers. The total counts of registered sections in the 10 fields were used to compare the distribution density of the nerves. Values were expressed as mean± SD . Statistical analyses were performed by analysis of variance (ANOVA) followed by Dunnett’s test or Student’s t -test. Differences were considered significant at P < 0.05. RESULTS 1 To determine the differences of cardiac sympathetic NET and myocardial β 1 -AR mRNA expression levels of LV between the 2- and 16-month-old rats, Northern blot was performed as previously described (Li et al., 2001 2001 ). The NET mRNA and protein levels were reduced in 16-month-old rats by 67% (Fig. 1 1 ) and 26% (Fig. 2 2 ), respectively, and β 1 -AR mRNA level in myocardia of the 16-month-old rats was reduced by 43% (Fig. 3 3 ) in comparison with that in the ganglia ( P =0.001) and that in myocardia ( P < 0.05, P =0.018) of 2-month-old rats, respectively. Meanwhile, about 51% reduction of the catecholaminergic nerve fiber density in the myocardia of 16-month-old rats was found (Fig. 4 4 ), compared with 2-month-old rats ( P < 0.05). * P < 0.05, 2-month-old rats vs 16-month-old rats; Mean± SD ; n =5 for each group * P < 0.05, 2-month-old rats vs 16-month-old rats; Mean± SD ; n =5 for each group * P < 0.05, 2-month-old rats vs 16-month-old rats. Mean± SD ; n =5 for each group * P < 0.05, 2-month-old rats vs 16-month-old rats; Mean± SD ; n =5 for each group DISCUSSION 1 Sympathetic nerve system (SNS) regulates the function of innervated effector organs through releasing and terminating neurotransmitter NE (Esler et al., 1990 1990 ; Francis, 1995 1995 ). The released transmitters in the cleft bind receptors on the postsynsptic membrane through diffusion, transmitting the signals to the postsynaptic neurons. After the signal transmission is completed, the released neurotransmitters in the cleft must be rapidly eliminated from the cardiac synaptic cleft primarily via the NET on presynaptic membrane, so as to assure effectiveness of the nerve impulse and control the strength of the nerve impulse in this way (Barkely et al., 1994 1994 ; Povlock and Amara, 1997 1997 ). The signal transmission termination of majority of monoamines and amino-acids neurotransmitters predominantly depends on the reuptake of neurotransmitter transporters. Eisenhofer ( 1994 1994 ) reported that 88% of released NE was removed by neuronal uptake, 4% was removed by extraneuronal uptake, and 8% escaped removal processes to spillover into plasma. Only a small fraction of sympathetic cleft NE gains access to the circulation, so that changes in transport or changes in reuptake could have a profound effect on the circulating levels. NET controls NE concentration in the cleft and around β-AR (Esler et al., 1990 1990 ; Barkely et al., 1994 1994 ; Eisenhofer, 1994 1994 ; Povlock and Amara, 1997 1997 ). It has long been recognized that prolonged exposure of myocardial tissue to β-adrenergic agonists modifies the β-AR responsiveness, and that densensitization of β-AR stimulation occurs via modifications of both receptor and postreceptor events. But evidence has accumulated that dysfunction of cardiac reuptake of NE leads to reduction of reuptake of NE and myocardial β 1 -AR with aging (Chevalier et al., 1991 1991 ; Dawson and Meldrum, 1992 1992 ; Sakai et al., 1992 1992 ; Lakatta, 1993 1993 ; White et al., 1994 1994 ; Esler et al., 1995 1995 ; Ferrara et al., 1997 1997 ; Hardouin et al., 1997 1997 ; Guimaraes et al., 1998 1998 ; Snyder et al., 1998 1998 ; Li et al., 2003 2003 ). Previous experimental results showed that NET mRNA was exclusively expressed in noradrenergic cells of the cardiac ganglia rather than in the myocardia (Ungerer et al., 1996 1996 ; Bohm et al., 1998 1998 ; Backs et al., 2001 2001 ; Li et al., 2001 2001 ), and was strictly localized to the perikarya but not detected in nerve fibers projecting from the ganglia (Backs et al., 2001 2001 ). The sympathetic origin of the cells stained positively for NET mRNA was specified by demonstration of TH immunofluorescence (Backs et al., 2001 2001 ). The results suggest that the NET proteins colocalized in the sympathetic nerve are transported from a distance from the heart; therefore it might also be considered as a marker of the noradrenergic nerve. But the distribution of NET proteins localized along the fibers in the myocardium may not be consistent with the TH positive fiber density. Recently, Wehrwein et al.( 2008 2008 ) reported that the NET protein is colocalized in nerve fibers with TH immunoreactivity in murine atria; however, the NET protein does not correlate with innervation density of murine heart after normalizing to the total protein content of each of the chambers. Rather, NET appears to be equally distributed throughout the myocardium; the total NET protein was highest in the ventricles and lowest in the atria (Wright et al., 2006 2006 ; Wehrwein et al., 2008 2008 ). In contrast, postsynaptic effector, β-AR, is uniformly distributed throughout the myocardium, with similar densities in both atrial and ventricular tissues (Hata et al., 2004 2004 ). The primary finding of present study is that age-related decline of NET levels of both mRNA and protein, together with the pattern of β 1 -AR mRNA expression, confirms the previous findings (Sakai et al., 1992 1992 ; White et al., 1994 1994 ; Ferrara et al., 1997 1997 ). They showed that myocardium decreased with aging, which is similar to cardiac sympathetic NET. Our results suggest that NET might contribute to the decrease in myocardial β 1 -AR mRNA with aging. The NET change appears accordant with previous observations (Shores et al., 1999 1999 ; Zhu et al., 2005 2005 ), which showed that NET mRNA expression in locus coeruleus (LC) was significantly decreased with aging in rats, and the level of NET in the LC of 23-month-old rats showed less than half of that found in the 1-month-old rats (Zhu et al., 2005 2005 ). These previous findings also indicate that there was an age-dependent decrease in myocardial NET binding sites by 36% in elderly patients [(60±3) years] without apparent heart failure, and NET activity was ~50% lower than that in children [(14±3) years] (Leineweber et al., 2002 2002 ). However, the present results showed that age-related reduction of NET mRNA appears not parallel to its protein expression level. This could be due to an increase in the rate of NET protein production or a decrease in NET protein removal, or both. Recent studies demonstrated that the ratios of protein levels do not have a one-to-one correlation with the ratios of the corresponding mRNAs, which suggests that the relation between transcription and translation, consequently between mRNA and protein, is complex (Ideker et al., 2001 2001 ; Mehra et al., 2003 2003 ). There is evidence indicating that the maximum rate of NE uptake (i.e., V max for uptake [ 3 H]NE) declined significantly and the number of NET per synaptosome may decline with age and decrease in β-AR density in rat heart (Snyder et al., 1998 1998 ). The reduction of the cardiac response to the sympathetic stimuli during aging may be partly explained by a decrease in the corresponding receptor density; the changes are reversible and the density of the receptors can return to adult control values by chronic administration of the appropriate antagonist (Chevalier et al., 1991 1991 ). Similarly, clinical studies showed that reduced NE reuptake increases the overflow of the neurotransmitter to plasma from the aging heart during stimulation of the cardiac sympathetic outflow (Esler et al., 1995 1995 ) and β-AR downregulation in the hearts of old subjects (White et al., 1994 1994 ). Furthermore, it is well documented that NET, the cardiac local modulator, is a major factor contributing to downregulation of myocardial β-AR density in heart failures of both human and animal models (Liang et al., 1989 1989 ; Bristow et al., 1992 1992 ). The important role of NET in terminating the action of released NE contributes to the maintenance of NE levels in the nerve terminals as well as to rapid regulation of NE levels around the receptors, which in turn influences the expression of post synaptic β-AR. In the present study, the changes of mRNA and protein levels suggested the lower transcription rather than NET internalization in the hearts of the aged rats, because changes in cell surface expression of transporter protein measured with antibody-based techniques, such as immunohistochemical and Western blot analyses, may not be detected. These techniques usually, but not always, measure the total number of transporters in the cell (Zahniser and Doolen, 2001 2001 ). Therefore, it is reasonable that NET protein biosynthesis reduces during aging, which likely contributes to the reduced cardiac reuptake capacity of NE. Since NET is a component of sympathetic nerves, the recent report by Bruzzone et al.( 2003 2003 ) of an age-related decrease of the sympathetic is consistent with the present finding of a lower NET density with age. The reduction in NET protein might also be at least partly due to a loss of the nerve terminals, even though the NET protein does not correlate with the innervation density in myocardium (Wright et al., 2006 2006 ; Wehrwein et al., 2008 2008 ). The finding suggests that the upstream modulator of β-AR, cardiac symapthetic NET, may have a predominant role in age-related density downregulation of myocardial β 1 -AR in senescent rats, which may contribute to both of β-AR downregulation and age-related decrease of cardiac β-adrenergic responsiveness. However, the mechanism responsible for downregulation of cardiac NET with aging is not clear at present; the reduction in mRNA could be due to a decrease in transcription of the gene and/or a change in mRNA stability. It is known that DNA binding activity of activating protein-1 transcription factor decreases with aging (Asanuma et al., 1995 1995 ; Tumer et al., 1997 1997 ), which could contribute to a lower transcription rate of the adenosine A-1 receptor (A1-AR) gene. Whether this is relevant to the expression of NET mRNA is not clear and will need further investigation. Moreover, there are an accumulating pool of evidence that the central neural mechanisms involved the increase of cardiac sympathetic nerve activity (Aggarwal et al., 2002 2002 ; Rosen et al., 2004 2004 ; Kaye and Esler, 2005 2005 ; Leenen, 2007 2007 ), so we conclude that alteration of central nervous system activity deteriorates significantly with aging, which might be implicated in the function of cardiac sympathetic nerve system. References A Aggarwal , MD Esler , GW Lambert , J Hastings , L Johnston , DM Kaye . Norepinephrine turnover is increased in suprabulbar subcortical brain regions and is related to whole-body. 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Age-associated changes in mRNA levels of Phox2, norepinephrine transporter and dopamine β-hydroxylase in the locus coeruleus and adrenal glands of rats . J Neurochem , 2005 . 94 ( 3 ): 828 - 838 . 16033425 DOI: 10.1111/j.1471-4159.2005.03245.x http://doi.org/10.1111/j.1471-4159.2005.03245.x . Age-associated changes in mRNA levels of Phox2, norepinephrine transporter and dopamine β-hydroxylase in the locus coeruleus and adrenal glands of rats The authors are indebted to Prof. Jian Fei of Tongji University, Shanghai, China for his valuable suggestions. 10.1631/jzus.B0820213 Project supported by the Postdoctoral Fellow Foundation of the Science and Technology Committee of Shanghai (No. 98-10) and the Natural Science Foundation of Chinese People’s Armed Police Force (Nos. WKH2006-5 and WKH2008ZO4), China 2008-07-10 2009-02-19 2008-12-01 2022-01-05 Copyright © 2009, Journal of Zhejiang University Science 2009 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 10