Cardiac Sympathetic Rejuvenation
A Link Between Nerve Function and Cardiac Hypertrophy
Neuronal function and innervation density is regulated by target organ-derived neurotrophic factors. Although cardiac hypertrophy drastically alternates the expression of various growth factors such as endothelin-1, angiotensin II, and leukemia inhibitory factor, little is known about nerve growth factor expression and its effect on the cardiac sympathetic nerves. This study investigated the impact of pressure overload-induced cardiac hypertrophy on the innervation density and cellular function of cardiac sympathetic nerves, including kinetics of norepinephrine synthesis and reuptake, and neuronal gene expression. Right ventricular hypertrophy was induced by monocrotaline treatment in Wistar rats. Newly developed cardiac sympathetic nerves expressing β3-tubulin (axonal marker), GAP43 (growth-associated cone marker), and tyrosine hydroxylase were markedly increased only in the right ventricle, in parallel with nerve growth factor upregulation. However, norepinephrine and dopamine content was paradoxically attenuated, and the protein and kinase activity of tyrosine hydroxylase were markedly downregulated in the right ventricle. The reuptake of [125I]-metaiodobenzylguanidine and [3H]-norepinephrine were also significantly diminished in the right ventricle, indicating functional downregulation in cardiac sympathetic nerves. Interestingly, we found cardiac sympathetic nerves in hypertrophic right ventricles strongly expressed highly polysialylated neural cell adhesion molecule (PSA-NCAM) (an immature neuron marker) as well as neonatal heart. Taken together, pressure overload induced anatomical sympathetic hyperinnervation but simultaneously caused deterioration of neuronal cellular function. This phenomenon was explained by the rejuvenation of cardiac sympathetic nerves as well as the hypertrophic cardiomyocytes, which also showed the fetal form gene expression.
Cardiac sympathetic nerves play an important role in regulating cardiac function, affecting heart rate, contractility, and conduction velocity. Moreover, the neurotransmitter of cardiac sympathetic nerves, norepinephrine (NE), induces cardiac hypertrophy. It is generally known that neuronal cellular function and innervation density are regulated by target organ-derived neurotrophic factors.1 Among them, the development and maintenance of the cardiac sympathetic and sensory nervous systems were controlled by cardiomyocyte-derived nerve growth factor (NGF).2 We recently reported that endothelin (ET)-1, but not angiotensin II, insulin-like growth factor-1, phenylephrine, or leukemia inhibitory factor, selectively upregulated NGF expression in cardiomyocytes via the ET-A receptor/Giβγ/protein kinase C–mediated signaling pathway.3 We also demonstrated that cardiac sympathetic nerve development was markedly retarded in ET-1–deficient mice because of the downregulation of NGF and, furthermore, that cardiac-specific overexpression of NGF in ET-1–deficient mice overcame the reduction in cardiac sympathetic innervation.3 These findings implicated ET-1/NGF signaling as critical for the development of cardiac sympathetic innervation.
Cardiac hypertrophy is an independent risk factor for heart failure and sudden cardiac death, with various neurohumoral factors and mechanical stretch involved in the process. Cardiac hypertrophy not only increases the cellular volume of cardiomyocytes but also induces fetal gene expression, including contractile proteins,4 ion channels,5 and secretory proteins, such as atrial natriuretic peptide6 and brain natriuretic peptide (BNP),7 which is explained as rejuvenation of cardiomyocytes. Mechanical stretch or pressure overload also upregulates hypertrophic growth factors and cytokines including ET-1.8 Stimulation of cultured cardiomyocytes with these factors also induces cellular hypertrophy and fetal gene expression.9 Although the effect of cardiac sympathetic nerves on cardiac hypertrophy has been described,10 little is known about the effect of cardiac hypertrophy on cardiac sympathetic nerves, including innervation density, kinetics of NE synthesis and reuptake, and neuronal gene expression. Previous studies revealed that ventricular pressure overload such as in pulmonary hypertension or transaortic coarctation induces cardiac hypertrophy and elevated ET-1 expression.8 Based on these observations and our findings, it is easy to speculate that a pressure overload elevates NGF expression via intrinsic ET-1 elevation, leading to hyperinnervation in the pressure-overloaded ventricle.
Regional sympathetic hyperinnervation may induce hypercontraction, increase conduction velocity, and, in some cases, enhance automaticity, which in turn causes imbalanced contraction and the potential for fatal arrhythmia.11 In the present study, we investigated the effect of cardiac hypertrophy on sympathetic hyperinnervation using a pulmonary hypertension-induced right ventricular (RV) hypertrophy (RVH) model and whether this anatomical hyperinnervation is associated with hyperfunction of cardiac sympathetic nerve systems. We unexpectedly found that pressure overload–induced cardiac hypertrophy not only caused a regional anatomical hyperinnervation but also resulted in a strong downregulation of neuronal cellular function and induction of fetal gene expression in sympathetic neurons. We therefore report for the first time that the pressure overload causes rejuvenation (hyperplasia, reduced NE uptake, reduced NE synthesis, and fetal gene expression) of the regional cardiac sympathetic nervous systems, as well as that of hypertrophic cardiomyocytes.
Materials and Methods
All experimental procedures and protocols were approved by the Animal Care and Use Committees of the Keio University and conformed to the NIH Guide for the Care and Use of Laboratory Animals.
Five-week-old male Wistar rats were treated with 60 mg/kg of monocrotaline (Sigma, St Louis, Mo) to produce pulmonary hypertension as described previously.12 The animals were euthanized on days 0, 10, 20, or 30. On the day of the experiment, rats were artificially ventilated under anesthesia with ketamine (60 mg/kg) and xylazine (15 mg/kg) given intraperitoneally. Thoracic aortic, left ventricular (LV), and RV pressures were recorded as described previously.13 After hemodynamic measurement, the rats were euthanized with pentobarbital and the hearts were removed. The hearts were divided into the RV and LV free wall, and each portion was weighed separately. Neonatal rats (P1 to P3) were also euthanized with pentobarbital and hearts were removed to be examined for immunohistochemical analysis.
Sample fixation, embedding, sectioning, and blocking were as described previously.14 To detect nerve fibers in the heart, frozen sections were incubated with single or double antibodies against tyrosine hydroxylase (TH) (CHEMICON, AB152; 1:200 and ImmunoStar, 22941; 1:1000), calcitonin gene–related protein (CGRP)15 (Biogenesis, 1720 to 9007; 1:200), β3-tubulin (COVANCE, PRB-435P; 1:2000), growth-associated protein (GAP43) (CHEMICON, AB5220; 1:4000 and CHEMICON, MAB347; 1:4000), and PSA-NCAM (the highly polysialylated neural cell adhesion molecule) (CHEMICON, MAB5324; 1:100). The following secondary antibodies were used: polyclonal swine anti-rabbit TRITC (DAKO; 1:200), Alexa Fluor 488–conjugated goat anti-mouse IgG (Molecular Probes; 1:200), Alexa Fluor 546–conjugated goat anti-mouse IgG (Molecular Probes; 1:200), Alexa Fluor 546–conjugated donkey anti-goat IgG (Molecular Probes; 1:200), and Cy3-conjugated donkey anti-mouse IgM (The Jackson Laboratory; 1:200). The samples were observed under a Zeiss LSM 510 META confocal microscopy (Oberkochen, Germany). Some sections for TH staining were visualized using the DAKO Liquid DAB Substrate–Chromogen System. Immunostained areas were quantified using NIH image, as described previously.11
The metaiodobenzylguanidine (MIBG) accumulation experiment was performed as described previously.17 Briefly, [125I]-MIBG (25 μCi/kg) was injected via the tail vein while the rats were under anesthesia with pentobarbital (30 mg/kg IP). Rats were euthanized at 3 hours after injection. Biodistribution of MIBG in the right ventricle, left ventricle, lung, spleen, and blood were measured with an automatic well-type gamma counter (ARC 2000, Aroka, Japan).
Evaluation of Uptake 1 Function
Cardiac [3H]-NE uptake was measured as described previously.17 [3H]-NE (25 μCi/kg) was injected intravenously under no treatment and under pretreatment with desipramine (10 mg/kg IP 1 hour before the intravenous injection of [3H]-NE). [3H]-NE uptake was measured 15 minutes postinjection in a liquid scintillation counter after tissue oxidation. The uptake 1 component was identified as the percentage difference in ventricular [3H]-NE uptake produced by pretreatment with desipramine compared with control values under no pretreatment. Uptake 1 function was evaluated by comparison of the uptake 1 component between the control and monocrotaline-injected rats.
Tissue Norepinephrine and Dopamine Measurement
Tissue samples were homogenized within 30 seconds in 0.1 N HCl containing 0.1% sodium pyrosulfite (Na2S2O5). After centrifugation (10 000g, 30 minutes), NE and dopamine were extracted with alumina and determined by high-performance liquid chromatography.
Assay for Tyrosine Hydroxylase Enzyme Activity
Assay for the TH enzyme activity was performed using high-performance liquid chromatography as described previously.18
Online Data Supplement
The methods of the other assays, including RNA extraction and poly(A)+RNA northern blotting, transmission electron microscopy, Western blot analysis, and ELISA for NGF, are described in the online data supplement.
Values are presented as means±SD. The significance of differences among mean values were determined by ANOVA. Statistical comparison of the control group with the treated group was performed using the nonparametric Fisher’s multiple comparison tests. The level accepted for significance was P<0.05.
Evaluation of RV Hypertrophy Induced by Monocrotaline
Transverse sections of the whole rat heart at 30 days (Figure 1A) showed that the RV free wall was markedly thickened and elongated and that the RV cavity was enlarged in monocrotaline-treated rats compared with untreated controls. RV systolic pressure reached 59±4 mm Hg at 30 days (Figure 1B), but LV end-systolic (data not shown) and end-diastolic pressure was not affected (Figure 1C). The ratio of RV weight to body weight increased 1.6-fold over the time compared with control rats (Figure 1D), whereas the LV weight to body weight ratio showed no significant change (Figure 1E). We had measured serum concentration of NE level in this experiment, but the serum level of NE was unchanged by the generation of PH in our model. These results indicated that monocrotaline treatment for 30 days is sufficient to induce RVH, and the following analyses were performed after exposure to monocrotaline for 30 days.
Pulmonary Hypertension Upregulates NGF in Hypertrophic Right Ventricles but Not in Left Ventricles
Northern blot analysis was performed to investigate whether RVH affects NGF expression similarly to ET-1 and cardiac stress marker BNP expression in the hypertrophic right ventricle. ET-1, NGF, and BNP gene expressions were rapidly increased by 9.4-, 6.3-, and 15-fold, respectively, in the right ventricle following induction of hypertrophy (Figure 1F). In contrast, NGF expression in the left ventricle was negligible, and the induction of BNP was less than that detected in the right ventricle. NGF protein was also increased by 4.8-fold in the right ventricle of monocrotaline-injected rats compared with controls, whereas levels were unchanged in the left ventricle (Figure 1G). These findings indicated that RVH induced by pulmonary hypertension increases NGF expression.
RV Hypertrophy Caused Hyperinnervation in the Right Ventricle
We next performed immunostaining for β3-tubulin, a neuronal marker expressed specifically in nerve fiber axons,19 and GAP43, a growth-associated cone marker protein expressed when the nerve terminal develops20 to investigate whether RVH-induced elevation of NGF could affect innervation density in the right ventricle. In control rats, β3-tubulin and GAP43 expression in both ventricles were observed at the epicardial area and sparsely at the intramyocardial layer (Figure 2A,B). In contrast, their expressions markedly increased in the hypertrophic right ventricles of monocrotaline-exposed rats (Figure 2A). Although newly developed thick nerves were prominent at the epicardial layer and the perivascular area, thin nerves were also detected in the right ventricular myocardium. By comparison, immunostaining for these proteins in the left ventricle was unaffected (Figure 2B). Quantitative analysis revealed an increase of 4.2-fold and 6.8-fold in the β3-tubulin– and GAP43-stained RV regions, respectively, of monocrotaline-exposed rats, compared with controls (Figure 2C and 2D). These findings indicated that RVH-induced hyperinnervation only in the right ventricle, concordant with the elevated NGF expression pattern.
Hyperinnervated Nerves Are Mainly Sympathetic Nerves
To determine whether the newly developed nerves were sympathetic or sensory, we performed double immunostaining for GAP43 and TH (a sympathetic neuron marker) or CGRP (a sensory neuron marker).2 GAP43-immunopositive neurons costained more frequently for TH than CGRP (Figure 3A), with 82% coimmunopositive for TH/GAP43 and 18% for CGRP/GAP43 (Figure 3B).
A more detailed morphological examination of the neurons was then undertaken by transmission electron microscopy. Figure 3C shows the axonal swellings of the nerve endings with many cored and noncored synaptic vesicles apposing the cardiomyocytes (arrow heads). Mitochondria and microtubules were observed in the axon, and part of the swelled axon is covered by the cytoplasm of surrounding Schwann cells. Figure 3D shows many axonal swellings (arrows) with some mitochondria and small vesicles surrounded by the cytoplasm of a single Schwann cell with surrounding external lamina, indicative of nerve sprouting. These findings were consistent with the ultrastructure of sympathetic nerves.21 Taken together, these results suggested that the hyperinnervated nerves in the hypertrophic right ventricle comprised mainly sympathetic neurons.
Paradoxical Decrease in Catecholamine Content and NE Reuptake Function in Hyperinnervated Right Ventricles
To investigate nerve cellular function of the RV anatomical hyperinnervation, we first measured the NE and dopamine content. Unexpectedly, both NE and dopamine in the hyperinnervated right ventricles were markedly decreased to 34% and 42% of the control (Figure 4A and 4B), respectively, whereas levels in left ventricles were unaffected (data not shown). To address a possible mechanism for this, we looked for excess secretion, reuptake dysfunction, or downregulation of NE production in the hyperinnervated right ventricles.
The density of [125I]-MIBG accumulation at 3 hours after injection reflects both the secretion level and reuptake function of NE. We administered [125I]-MIBG to both control and treated rats and measured their accumulation rates 3 hours later. Autoradiography of heart slices taken in short-axis view clearly showed a significantly lower density in the right ventricle compared with the left ventricle in monocrotaline-exposed rats, whereas no significant difference was observed between ventricles in the control group (Figure 4C and 4D).
To assess whether the reduced MIBG accumulation was attributable to excess secretion or reuptake dysfunction of NE, we measured the reuptake function (uptake 1) at sympathetic nerve terminals. Uptake 1 function of [3H]-NE in hyperinnervated right ventricle was significantly decreased compared with the control group (Figure 4E). These findings indicated that the reduced MIBG accumulation observed in hyperinnervated right ventricle was caused by downregulation of NE reuptake function.
Norepinephrine Synthesis Was Markedly Decreased in the Hyperinnervated Right Ventricle
Next, we investigated whether NE synthesis was downregulated in hyperinnervated right ventricle. TH is an enzyme that catalyzes the conversion of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-DOPA) and is a rate-determining enzyme for catecholamine synthesis. TH protein expression was markedly attenuated in monocrotaline-treated right ventricles by 21% of the controls (Figure 5A and 5B). The proportion of the hyperinnervated right ventricles immunostained for TH was significantly decreased to 53% of that in controls (Figure 5C and 5D), whereas LV staining for TH was not different between treated and control rats unaffected (data not shown).
We further investigated TH enzyme activity in this model by measuring l-DOPA production in myocardial lysate. The TH enzyme activity of the hyperinnervated right ventricles was markedly decreased to 35% of activity in the control animals (Figure 5E). Because these methods did not directly measure NE secretion, it is possible that excess secretion of exogenous NE influenced the catecholamine contents in the beginning of hypertrophic right ventricles. Nevertheless, these findings indicated that the decrease in NE and in its upstream substrate dopamine were caused by the downregulation of both NE reuptake function and catecholamine synthetic enzyme, regardless of the sympathetic hyperinnervation in the stressed right ventricle.
Immature and Fetal Neuron Marker Was Expressed in Hypertrophic Right Ventricle
To explain the paradox between anatomical sympathetic hyperinnervation and the functional deterioration in a stressed heart, we investigated the possibility that the anatomical hyperinnervated nerve takes the fetal or immature form. We performed immunostaining for PSA-NCAM, a known marker of immature neurons.22 A number of PSA-NCAM–immunopositive neurons costained with GAP43 in hypertrophic right ventricle of the monocrotaline-exposed rats (Figure 6A). PSA-NCAM was clearly expressed in the rat neonate heart, but not in the control adult heart (Figure 6B). Interestingly, PSA-NCAM was strongly expressed in the hyperinnervated right ventricle. Quantitative analysis revealed that the right ventricle of monocrotaline-exposed rats showed a 16-fold increase in area of PSA-NCAM staining compared with controls (Figure 6C). Western blot analysis confirmed that PSA-NCAM was strongly expressed in the right ventricles of monocrotaline-exposed rats, but not of control untreated rats (Figure 6D).
Together, the findings presented indicated that cardiac hypertrophy induces anatomical hyperinnervation, but that these nerves express a fetal or immature phenotype with decreased synthesis and reuptake of NE.
We demonstrated herein that PH induced ET-1 and NGF elevation specifically in the right ventricle, which might be associated with sympathetic hyperinnervation in a chamber-specific manner. Surprisingly, these anatomically hyperinnervated sympathetic neurons showed markedly decreased NE synthesis via downregulation of TH, as well as an attenuated NE reuptake, leading to depletion of NE and dopamine content. The hyperinnervated sympathetic neurons strongly expressed immature neuron-specific marker PSA-NCAM, indicating neuron rejuvenation, and providing a possible explanation for the dissociation of anatomical hyperinnervation and functional depression.
Zhou et al23 reported that cardiac nerve sprouting and sympathetic hyperinnervation occurred after myocardial infarction, and that this may have been caused by local NGF release. Backs et al24 found that impaired NE reuptake, depleted NE stores, and reduced NE synthesis were induced after aortic banding, without loss of nerve terminals. These reports strongly support our observations.
Cardiac Hypertrophy Activates ET-1/NGF Pathway and Causes Sympathetic Hyperinnervation
We previously demonstrated in cultured neonatal cardiomyocytes and ET-1 gene–targeted mice that ET-1 specifically increases NGF expression in cardiomyocytes and that ET-1/NGF signaling is critical for sympathetic nerve development.3 It is also well known that mechanical stretch in vitro and pressure overload in vivo enhance ET-1 expression in cardiomyocytes.8 The present study revealed that cardiac hypertrophy upregulated the ET-1/NGF signaling pathway, with the possible effect of inducing sympathetic hyperinnervation in adult animal models. This suggested that this pathway is pivotal not only for sympathetic nerve development but also for its regulation in the adult. However, because various growth factors and cytokines are elevated in pressure-overloaded cardiac hypertrophy, it is possible that other factors and/or mechanical stretch itself are involved in the NGF upregulation. Future delineation of upstream factors regulating these changes in NGF expression is therefore needed.
In the present study, we investigated the expression of NGF and the anatomical and functional condition of sympathetic neurons in cardiac hypertrophy. Kaye et al25 and Qin et al26 have reported that cardiac NGF is downregulated in end-stage heart failure. Thus, it is important to distinguish the condition of sympathetic nerves at the stage of cardiac hypertrophy or compensated heart failure from that at the stage of terminal decompensated heart failure.
Sympathetic Hyperinnervation and Functional Deterioration Accompanying the Nerve Rejuvenation
GAP43 is a marker for neuronal growth cones.20 Expression of GAP43 at the anatomically hyperinnervated sympathetic nerves therefore indicated some nerve sprouting to hypertrophic cardiomyocytes. PSA-NCAM is a marker of immature neurons, and is usually expressed in developmental stage neurons or regenerated neurons from neural stem cells. The expression of PSA-NCAM at the cell surface is developmentally regulated and plays an important role in several developmental events such as cell migration,27 neurite outgrowth,28 and nerve branching29 in both the developing and adult nervous system. Expression of PSA-NCAM in the adult brain is suggestive of the maintenance or reappearance of a developmental state.22,30 Conversely, increased PSA-NCAM has also been observed during reinnervation after injury in peripheral nerves.31,32 Our findings indicated that the hyperinnervated sympathetic nerves observed in this study were rejuvenating neurons, showing neurite outgrowth and nerve branching over the hypertrophic cardiomyocytes.
We recently reported that cardiac sensory nerves express CGRP and that their innervation density was regulated by cardiomyocyte-derived NGF.2 The present findings of CGRP/GAP43-positive neurons could be explained by this mechanism. Because the CGRP receptor is expressed in the stellate ganglion33 and CGRP itself is a regulator of sympathetic nerves, it is possible that CGRP played a role in mediating the sympathetic nerve changes that we observed.
Various studies have shown hypertrophic cardiomyocytes expressing a fetal phenotype gene pattern (rejuvenation). The phenotypic changes in hypertrophic cardiomyocytes include contractile proteins,4 ion channels,5 Ca2+-regulated proteins such as SERCA-2a,34 and energy production molecules,35 which cause the depressed function including decreased contraction and relaxation velocity and lowered ATP production. Although the precise mechanisms underlying these changes remain unclear, autocrine/paracrine factors from cardiomyocytes are possible players in this rejuvenation. Based on our study, we propose that rejuvenation of sympathetic nerves might play a crucial role in the depressed neuronal function of NE synthesis and reuptake, and further studies are required to address this hypothesis.
Molecular Mechanism of the Dissociation of Sympathetic Hyperinnervation and Depressed Neuronal Cellular Function
It seems paradoxical that the anatomical sympathetic hyperinnervation and depressed neuronal function may occur simultaneously. However, considering that this phenomenon was accompanied with neuron rejuvenation, it might not actually be a specific effect, but instead common to other cell types. Firstly, smooth muscle cells have both proliferative and contractile phenotypes and switch to a fetal or regenerating gene expression pattern in the proliferative phenotype.36 This is paralleled by cardiomyocytes responding to various stimuli with cardiac hypertrophy. If this is the case with sympathetic neurons, it is easy to understand the phenotypic changes observed here, including the depressed neuronal function and rejuvenation state.
Secondly, various growth factors and cytokines were induced in the pressure-overloaded cardiomyocytes, and these factors are known stimulators of hypertrophy and a fetal gene-expression switch in cardiomyocytes.10 The present study demonstrated that hyperinnervation and rejuvenation occurred in a pressure-overloaded and chamber-specific manner. Other studies have shown that externally administered NGF augments NE reuptake and sympathetic transmission.37,38 Although the hyperinnervation that we observed can be explained by upregulation of NGF, it is possible that some of the other factors elevated in pressure-overloaded cardiomyocytes were responsible for the sympathetic neuron rejuvenation and that local tissue-derived factors may have stimulated the presynaptic neuronal changes.
Thirdly, it is possible that a negative-feedback mechanism might underlie the downregulation of neuronal function to prevent excess signaling from the hyperinnervated sympathetic nerves. ET-1, which was elevated in this model, inhibits the neuronal norepinephrine transporter in the heart, suggesting a downregulation of neuronal function.39 Further investigations will be required to determine whether other factors are involved in this process.
Significance of This Phenomenon From a Clinical Viewpoint
Congestive heart failure (CHF) always accompanies cardiac hypertrophy from the subclinical stage. Myocardial NE content is decreased in CHF, and this phenomenon is explained by the concept that excess secretion of NE causes its depletion.40 Our findings could account for the decreased myocardial NE content in CHF; however, an oversecretion of NE does not explain our findings of decreased dopamine content, downregulation of NE reuptake, or decreased TH expression.
Many studies demonstrated a depressed MIBG reuptake in severe CHF, and this was attributed to cardiac sympathetic nerves being denervated.41 There are also reports of decreased NGF expression in end-stage heart failure, with induction of cardiac sympathetic denervation.25,26 Partial denervation could therefore explain the decreased MIBG uptake and NE content observed in our experiments. However, the precise pathological, functional, and molecular analyses could not be performed in the clinical situation. Our findings also could explain this phenomenon; both the cardiac denervation and the rejuvenation of sympathetic nerves might result in a similar phenotype, and some of these conditions might coexist in the clinical setting.
Future studies should aim to clarify whether (1) rejuvenation and hyperinnervation of sympathetic nerves in cardiac hypertrophy is reversible, (2) the rejuvenated and hyperinnervated neurons may progress to cardiac denervation if the cardiac hypertrophy leads to CHF, and (3) the rejuvenation and hyperinnervation is beneficial or harmful to cardiac hypertrophy or CHF. We also want to know how β-blocker therapy may affect these processes in sympathetic nerves. The relationship between sympathetic rejuvenation and the regulation of regional electrical and mechanical function at the various stages of RVH should also be determined. Despite its importance, the present study did not investigate the parasympathetic nervous systems in the RVH model. In addition, the status of the parasympathetic nervous system in the RVH model remains to be elucidated.
In conclusion, to our knowledge, this is the first report that rejuvenation of sympathetic neurons can be induced by the postsynaptic environment (cardiac hypertrophy) and can be associated with hyperinnervation, decreased NE synthesis, and depressed reuptake of NE. NGF is clearly pivotal in this process, but it remains unknown what other factors are involved. Further studies are required to understand this phenomenon and its precise mechanism.
Sources of Funding
This study was supported in part by research grants from the Moritani Scholarship Foundation; the Ministry of Education, Culture, Sports, Science and Technology, Japan; and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation.
Original received December 30, 2006; resubmission received April 4, 2007; revised resubmission received April 27, 2007; accepted May 2, 2007.
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