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Mechanical stretch induces nerve sprouting in rat sympathetic neurocytes
Autonomic Neuroscience: Basic and Clinical 155 (2010) 25–32
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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Mechanical stretch induces nerve sprouting in rat sympathetic neurocytes Obaida R. Rana a,⁎, Patrick Schauerte a, Dorothee Hommes a, Robert H.G. Schwinger b, Jörg W. Schröder a, Rainer Hoffmann a, Erol Saygili a a b
Department of Cardiology, RWTH Aachen University, Aachen, 52074, Germany Medical Clinic II, Klinikum Weiden, Weiden, 92637, Germany
a r t i c l e
i n f o
Article history: Received 10 October 2009 Received in revised form 30 December 2009 Accepted 5 January 2010 Keywords: Sympathetic nerve sprouting SCG Mechanical stretch Heart failure Myocardial infarction NGF CNTF
a b s t r a c t Sympathetic nerve sprouting (SNS) has been shown to occur after myocardial infarction (MI) and heart failure (HF) and is known to be responsible for the development of lethal arrhythmias. During MI or HF intracardiac cells are exposed to increased mechanical stretch. Molecular mechanisms which trigger sympathetic neural growth are largely unknown. Therefore, this study aimed to investigate the impact of mechanical stretch on rat neonatal sympathetic neurocytes of the superior cervical ganglion (SCG). Mechanical stretch resulted in an increased growth of sympathetic neurocytes. Furthermore, we could demonstrate that SCG neurocytes express nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3) and glial derived neurotrophic factor (GDNF) on mRNA and protein level. An increased NGF and CNTF expression, a down-regulated GDNF expression and an unchanged NT-3 expression were identified in the neurocyte cell culture supernatant of neurocytes exposed to mechanical stretch. However, neither brain derived neurotrophic factor (BDNF) mRNA and protein was expressed in SCG neurocytes, nor BDNF could be detected in the cell culture supernatant of SCG neurons. By anti-neurotrophin neutralizing experiments NGF and CNTF were identified as important stretch-induced growth-inducing factors. Losartan, an angiotensin-II type 1 receptor inhibitor, abolished the stretch-induced increase of NGF and CNTF expression and thereby prevented the stretch-induced neural growth. This study provides new molecular mechanisms by which the inhibitory effect of angiotensin-II type 1 receptor blockers on the neural/arrhythmogenic remodeling can be explained. However, further in-vivo studies are required to address this important issue. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Sympathetic nerve sprouting (SNS) is a major factor for the development of malignant arrhythmias after myocardial infarction (MI) (Zhou et al., 1995) and congestive heart failure (HF) (Himura et al., 1993). Pathological outgrowth of sympathetic fibers and subsequent hyperinnervation seems to be an important contributor to an elevated sympathetic tone and has been reported to occur within the first hours of MI (Oh et al., 2006). Furthermore, SNS has been suggested to be a major contributor to sudden cardiac death (Cao et al., 2000a; Cao et al., 2000b). After MI increased nerve growth factor (NGF) expression has been shown to promote sympathetic hyperinnervation (Hasan et al., 2006), and may represent an adaptive mechanism by which the heart tries to maintain ventricular contractility at the cost of eventually triggered ventricular arrhythmias. There is evidence that inflammatory cells like myofibroblasts and macrophages contribute to the development of SNS in the ischemic injury area after MI (Hasan et al., 2006). If ⁎ Corresponding author. Department of Cardiology, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany. Tel.: +49 241 8035142; fax: +49 241 8082482. E-mail address: [email protected] (O.R. Rana). 1566-0702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2010.01.003
other contributing factors than inflammatory cells or NGF are involved in the pathogenesis of SNS still needs to be investigated. Mechanical stretch is such condition to which the cardiac cells are exposed after MI and during HF (Zimmerman et al., 2000; Ashikaga et al., 2005; Gao et al., 2007; Koba et al., 2008). Recently, we could demonstrate that mechanical stretch of ventricular cardiomyocytes induces a significant down-regulation of NGF (Rana et al., 2009). However, a stretch-induced NGF down-regulation in cardiomyocytes would not explain the induction of SNS after MI and HF. Because the myocardium in extensively innervated by the sympathetic nervous system and the impact of mechanical stretch on sympathetic neurocytes is unknown, this study aimed to investigate the impact of mechanical stretch on rat neonatal sympathetic neurocytes of the superior cervical ganglion (SCG). Furthermore, stretch-induced alterations of the neurotrophins NGF, neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), glial derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) were investigated. Mechanical stretch resulted in increased neural growth assessed by measuring the neurite outgrowth and growth associated protein 43 (GAP-43) expression. Furthermore, in the cell culture supernatant of neurocytes exposed to mechanical stretch NGF and CNTF expression was significantly increased, GDNF expression was down-regulated, NT-3 expression was unchanged,
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while BDNF could not be detected. By anti-neurotrophin neutralizing experiments, NGF and CNTF were identified as important stretchinduced neural growth-inducing factors. Angiotensin-II type 1 (AT-1) receptor blockade with losartan completely prevented the stretchinduced neural growth by attenuating the stretch-induced increase of NGF and CNTF. 2. Materials and methods 2.1. Superior cervical ganglia (SCG) neurocyte cell culture All animals were cared for according to institutional animal-care requirements. Primary cultures of SCGs were performed from postnatal day 1 to 3 Sprague Dawley rats (Charles River, Germany) as described previously (Rana et al., 2009). Briefly, both SCG of 20–30 neonatal rat were dissected from the carotid bifurcation, cut into small pieces, and then transferred into 1 ml of Neuronal Base Medium (PAA, U15-024) supplemented with collagenase II (1.0 mg/ml, 280 U/ mg, Biochrom, C2-22), trypsin (0.5 mg/ml, trypsin 1:250, PAA, L-11-
002) and 1% penicillin–streptomycin (Sigma, P4458). Enzymatic digestion was performed for 40 min at 37 °C. Thereafter, SCGs were dissociated mechanically with fire-polished glass pipettes and centrifuged at 3000 rpm for 2 min. Cells were resuspended in 5 ml Neuronal Base Medium supplemented with 10% fetal bovine serum (FBS, PAA, A-15-101) and 1% penicillin–streptomycin. Cells were incubated for 2 h in an incubator (95% air, 5% CO2, 100% humidity) to allow non-neuronal cell (e.g. fibrocytes and Schwann cells) to attach, while neuronal cells need longer incubation duration to attach. After 2 h of incubation cell supernatant was centrifuged at 3000 rpm for 2 min and cells were resuspended in final Neuronal Base Medium supplemented with 10% FBS, 1% penicillin–streptomycin, 2 mM Lalanyl–L-glutamine (Gibco Invitrogen Corporation) and 10 µM 5Bromo-2′-deoxy-uridine (5-BrdU, Sigma, B-9285). Cells were plated onto silicone membranes coated with collagen type I (Bioflex Collagen I 6-well plates, Dunn, BF-3001C) at a density of 100,000 cells per well. With this procedure we could achieve purity of 93% of tyrosine hydroxylase (TH) positive neuronal cells (3 cell preparations, 635 cells of total 684 cells were TH positive by immunofluorescence, data
Fig. 1. Mechanical stretch induces growth of rat neonatal sympathetic neurocytes. (A) Measurements of neurite outgrowth in µm, n = 3 preparations each, *p < 0.01 vs. stretch 0 day, (B) net increase of neurite outgrowth in µm, n = 3 preparations each, *p < 0.01 vs. stretch, (C) representative and quantitation of GAP-43 Western blots, n = 3 preparations, *p < 0.01 vs. stretch, (D) representative images of control and stretched neurocytes in cell culture, scale bar 20 µm, and (E) representative tyrosine hydroxylase (TH) and growth associated protein 43 (GAP-43) immunofluorescence images of stretched neurocytes demonstrating the sympathetic origin of the cultivated neurocytes.
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(steps: 3, 7, 13 %) (Rana et al., 2009). Un-stretched control neurocytes were treated equally without application of mechanical stretch. 2.3. Antibodies and neurotrophin neutralizing antibodies
Fig. 2. mRNA expression of NGF, CNTF, NT-3 and GDNF in neonatal rat SCG neurocytes. Real-time PCR experiments of SCG neurocytes were performed after 72 h of stretch application. Stretch resulted in a significant up-regulation of NGF and CNTF mRNA (n = 3 preparations each). The mRNA expression of NT-3 was unchanged in stretched neurocytes compared to un-stretched control neurocytes (n = 3 preparations each). The gene expression of GDNF was significantly down-regulated in stretched neurocytes (n = 3 preparations each). The BDNF gene expression in control and stretched SCG neurocytes was not detectable, *p < 0.02 vs. control.
not shown). During the first 48 h SCG neurocytes were grown in serum-containing medium. Thereafter, stretch was introduced to attached neurocytes by applying a gradual increase of tension over a period of 72 h in a complete serum-free condition.
2.2. Application of homogeneous equibiaxial static stretch Static stretch was introduced to attached neurocytes by applying a gradual increase of tension as described previously (Rana et al., 2008). Because myocardium dilatation in most cases is a chronic adaptive response of cells to mechanical load neurocytes were exposed to a gradual increase of stretch every 24 h from 3 to 13% over 3 days
Following primary antibodies were used: rabbit anti-NGF (Santa Cruz, sc-548), rabbit anti-NT-3 (Santa Cruz, sc-547), rabbit anti-BDNF (Santa Cruz, sc-546), goat anti-CNTF (Santa Cruz, sc-1912), rabbit anti-GDNF (Santa Cruz, sc-328), rabbit anti-GAP-43 (Santa Cruz, sc10786), rabbit anti-tyrosine hydroxylase (Santa Cruz, sc-14007) and rabbit anti-GAPDH (Cell Signaling, #2118). Neurotrophin neutralizing antibodies: anti-NGF (Sigma-Aldrich, #N6655, 1:500 dilution), anti-CNTF (R&D Systems, #MAB557, 5 µg/ml), anti-GDNF (R&D Systems, #MAB212, 5 µg/ml) and anti-NT-3 (Promega, #G1651, 5 µg/ml). 2.4. RNA preparation, first-strand cDNA synthesis and quantitative realtime reverse transcription-PCR RNA extraction, first-strand cDNA synthesis and quantitative real-time PCR experiments were performed as described previously (Rana et al., 2008). Each preparation contained sufficient mRNA concentration for first-strand cDNA synthesis. PCR primers and fluorogenic probes for NGF, CNTF, GDNF, NT-3, BDNF and the endogenous control were purchased from Applied Biosystems (Foster City, CA). The assay numbers were as follows: Rn01533872_m1 (Ngfb), Rn00755092_m1 (Cntf), Rn00569510_m1 (Gdnf), Rn00579280_m1 (Ntf3), Rn00560868_m1 (Bdnf), and Rn00560865_m1 (beta-2 microglobulin). 2.5. Neurite outgrowth To measure the net increase of neurite outgrowth of SCG neurocytes, 10 light-optical microscopic images (200× magnification) were taken
Fig. 3. Neonatal rat SCG neurocytes express NGF, CNTF, NT-3 and GDNF. Representative immunofluorescence images of NGF, CNTF, NT-3 and GDNF in cultured neonatal rat SCG neurocytes. No BDNF-immunostaining could be revealed in cultured neonatal rat SCG neurocytes (image not shown).
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before (0 h) and after (72 h) the application of mechanical stretch of each preparation. The mean length of all neurocyte cell extensions was measured with the help of a scale bar on each microscopic image. The net increase of neurite outgrowth was assessed by subtraction of the mean neurite outgrowth after 72 h of stretch from the mean neurite outgrowth before stretch application. For each condition at least 3 cell preparation were performed with 1 well (100,000 cells). Number of neurocytes considered for neurite outgrowth measurement before/after stretch application: 641(before)/518(after) control cells (10 preparations), 603/479 stretched cells (10 preparations), 181/137 stretched cells + anti-NGF (3 preparations), 170/143 stretched cells + anti-NT-3 (3 preparations), 176/155 stretched cells + anti-CNTF (3 preparations), 199/135 stretched cells+ anti-GDNF (3 preparations) and 197/147 stretched cells + losartan (3 preparations). 2.6. ELISA, Western blot and immunofluorescence At 0, 24 and 72 h, the conditioned medium was collected and assayed. NGF, CNTF, GDNF, NT-3 and BDNF were assessed by ELISA kits from R&D Systems (Minneapolis, USA) according to the manufacturer's protocol. Western blot and immunofluorescence experiments were performed as described previously (Rana et al., 2009; Saygili et al., 2009). Cell lysis of each SCG preparation contained sufficient protein concentration to perform Western blot experiments for GAP-43 and GAPDH detection.
Figs. 3 and 5 were analyzed by 2-way-ANOVA followed by Bonferroni's post-hoc test. p-values <0.05 were considered as statistically significant. 3. Results 3.1. Mechanical stretch induces growth of neonatal rat sympathetic neurocytes Mechanical stretch of SCG neurocytes over a time period of 72 h resulted in a significant increase of neurite outgrowth compared to control neurocytes (in µm: control: 0 h = 41.9 ± 1.3 vs. 72 h = 49.4 ±2.4, p=0.13, n=3 preparations; stretch: 0 h=45.1±0.9 vs. 72 h=71± 1.7, p<0.01, n=3 preparations) (Fig. 1A). The net increase of neurite outgrowth of stretched neurocytes after 72 h was significantly increased compared to un-stretched control neurocytes (control: 7.5±1.7 vs. stretch: 25.9±1.3, n=3 preparations, p<0.01) (Fig. 1B). Furthermore, the GAP-43 protein expression, a marker for neural growth, was significantly increased in stretched neurocytes compared to un-stretched control neurocytes (control: 1±0 vs. stretch: 1.35±0.12, n=3 preparations each, p<0.01) (Fig. 1C). Fig. 1D demonstrates representative images of control and stretched neurocytes in cell culture. To demonstrate the sympathetic origin of our cultivated neurocytes, tyrosine hydroxylase (TH) and GAP-43 immunofluorescence experiments were performed on stretched neurocytes (Fig. 1E). 3.2. Neonatal rat SCG neurocytes express NGF, CNTF, NT-3 and GDNF mRNA
2.7. Statistical analysis All values are expressed as mean±SEM. Comparisons of two groups were performed by Student's t-test and multiple groups presented in
Next, we performed real-time PCR experiments of SCG neurocytes after 72 h of stretch application. The expression of the neurotrophins NGF, NT-3, GDNF, CNTF and BDNF was analyzed (Fig. 2). Stretch resulted in a
Fig. 4. Mechanical stretch results in up-regulation of NGF and CNTF and down-regulation of GDNF, while NT-3 demonstrates an unchanged protein expression pattern. (A,B) Mechanical stretch resulted in a significant up-regulation of NGF and CNTF concentration in the cell culture supernatant of stretched sympathetic neurocytes compared to un-stretched control neurocytes after 24 and 72 h assessed by ELISA. #p < 0.01 vs. control 24 h, *p < 0.01 vs. control 72 h. (C) The expression of GDNF was significantly down-regulated in stretched neurocytes after 72 h. *p < 0.01 vs. control 72 h (D) Mechanical stretch showed no significant effect on the NT-3 expression compared to un-stretched control neurocytes.
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significant up-regulation of NGF and CNTF mRNA in stretched neurocytes (NGF: control 1±0 vs. stretch 1.2±0.01, n=3 preparations each, p<0.02; CNTF: control 1±0 vs. stretch 1.43±0.03, n=3 preparations each, p < 0.02). The mRNA expression of NT-3 was unchanged in stretched neurocytes compared to un-stretched control neurocytes (NT-3: control 1 ± 0 vs. stretch 0.96 ± 0.05, n = 3 preparations each, p = 0.65). Furthermore, the gene expression of GDNF was significantly down-regulated in stretched neurocytes (GDNF: control 1 ± 0 vs. stretch 0.78 ± 0.01, n = 3 preparations each, p < 0.01). However, the BDNF gene expression in control and stretched SCG neurocytes was not detectable. 3.3. Detection of NGF, CNTF, NT-3 and GDNF protein expression in neonatal rat SCG neurocytes by immunofluorescence To investigate if neonatal rat SCG neurocytes express neurotrophins on protein level, NGF, CNTF, NT-3, GDNF and BDNF were analyzed by immunofluorescence in un-stimulated neurocytes. The purity of our cell culture was 93% as described in the Materials and methods section. By immunofluorescence experiments we could demonstrate that SCG neurocytes express NGF, CNTF, NT-3 and GDNF (Fig. 3). However, BDNF protein expression could not be revealed (image not shown).
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tions each) (Fig. 5A). In analogy to the neurite outgrowth measurements, stretch resulted in a significant increase in GAP-43 protein expression. NT-3 or GDNF neutralizing antibodies showed no effect on the stretch-induced increase in GAP-43 protein expression. However, NGF or CNTF neutralizing antibodies completely abolished the stretchinduced increase of GAP-43 protein expression (control 1 ± 0, stretch 1.6 ± 0.1, anti-NT3 1.6 ± 0.1, anti-GDNF 1.7 ± 0.1, anti-NGF 1 ± 0.1, antiCNTF 1.1 ± 0.1, n = 3 preparations each) (Fig. 5B). 3.6. Losartan prevents the stretch-induced alterations of neurotrophins In order to investigate the impact of AT-1 receptor inhibition on the stretch-induced growth response, neurocytes were treated with losartan (1×10− 6 mol/l) during mechanical stretch. Losartan completely prevented the stretch-induced up-regulation of NGF and CNTF in the cell culture supernatant (NGF: 0 h: control 1±0 vs. stretch+losartan 1±0; 24 h: control 1.02±0.01 vs. stretch+losartan 1.02±0.01, p=0.34; 48 h: control 1.1 ± 0.01 vs. stretch+ losartan 1.13± 0.03, p = 0.93; n = 8 preparations each) (CNTF: 0 h: control 1±0 vs. stretch+losartan 1±0; 24 h: control 0.99±0.01 vs. stretch+losartan 0.97±0.01, p=0.29; 48 h: control 1.02±0.01 vs. stretch+losartan 0.96±0.01, p=0.17; n=8
3.4. Mechanical stretch results in up-regulation of NGF and CNTF and down-regulation of GDNF, while NT-3 demonstrates an unchanged protein expression pattern Mechanical stretch resulted in a significant up-regulation of NGF and CNTF concentration in the cell culture supernatant of stretched sympathetic neurocytes compared to un-stretched control neurocytes after 24 and 72 h assessed by ELISA (NGF: 0 h: control 1±0 vs. stretch 1± 0; 24 h: control 1.02±0.01 vs. stretch 1.16±0.01, p<0.01; 48 h: control 1.1±0.01 vs. stretch 1.35±0.01, p<0.01; n=8 preparations each) (CNTF: 0 h: control 1±0 vs. stretch 1±0; 24 h: control 0.99±0.01 vs. stretch 1.2±0.01, p<0.01; 48 h: control 1.02±0.01 vs. stretch 1.41± 0.01, p<0.01; n=8 preparations each) (Fig. 4A and B). The expression of GDNF was significantly down-regulated in stretched neurocytes after 72 h (GDNF: 0 h: control 1±0 vs. stretch 1±0; 24 h: control 1.04±0.01 vs. stretch 0.83±0.02, p=0.07; 48 h: control 1.18±0.02 vs. stretch 0.69± 0.03, p<0.01; n=8 preparations each) (Fig. 4C). However, mechanical stretch demonstrated no significant effect on NT-3 expression as compared to un-stretched control neurocytes (NT-3: 0 h: control 1±0 vs. stretch 1±0; 24 h: control 0.97±0.01 vs. stretch 0.96±0.01, p=0.82; 48 h: control 0.94±0.01 vs. stretch 0.9±0.01, p=0.29; n=8 preparations each) (Fig. 4D). In analogy to the gene expression results, the neurocyte cell culture supernatant was free of BDNF. By 2-way-ANOVA analysis the means of values for 24 h and 72 h of NGF and CNTF demonstrated a statistical significance (p<0.05) compared to the means of 0 h. Furthermore, the means of 24 h were statistical significant as compared to 72 h for NGF and CNTF. GDNF and NT-3 demonstrated nonsignificant mean values as compared to all 3 time points. 3.5. NGF and CNTF are important stretch-induced sympathetic neurocyte growth-inducing neurotrophins To investigate which neurotrophic factors are involved in the stretchinduced neural growth of rat sympathetic neurocytes, stretched neurocytes were co-treated with anti-neurotrophin neutralizing antibodies and the net increase of neurite outgrowth (Fig. 5A) and GAP-43 protein expression (Fig. 5B) experiments were performed. NGF or CNTF neutralizing antibodies abolished the stretch-induced increase of neurite outgrowth, while NT-3 or GDNF neutralizing antibodies had no influence on the stretch-induced increase of neurite outgrowth (in µm: control 7.5 ± 1,7, stretch 25.9± 1.3, anti-NT-3 24.2 ± 1.0, antiGDNF 27.6 ± 1.4, anti-NGF 6 ± 2.8, anti-CNTF 13.7 ± 0.4, n = 3 prepara-
Fig. 5. NGF and CNTF are important stretch-induced sympathetic neurocyte growthinducing neurotrophins. (A) Stretched neurocytes were co-treated with anti-neurotrophin neutralizing antibodies and the net increase of neurite outgrowth was assessed. NGF or CNTF neutralizing antibodies abolished the stretch-induced increase of neurite outgrowth, while NT-3 or GDNF neutralizing antibodies had no influence on the stretch-induced increase of neurite outgrowth (n = 3 preparations each, #p < 0.05 vs. control, *p < 0.05 vs. stretch). (B) Stretch resulted in a significant increase in GAP-43 protein expression. NT-3 or GDNF neutralizing antibodies showed no effect on the stretch-induced increase in GAP-43 protein expression. NGF or CNTF neutralizing antibodies completely abolished the stretch-induced increase of GAP-43 protein expression (n = 3 preparations each, #p < 0.05 vs. control, *p < 0.05 vs. stretch).
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preparations each) (Fig. 6A and B). Furthermore, losartan abolished the stretch-induced down-regulation of GDNF (GDNF: 0 h: control 1±0 vs. stretch+losartan 1±0; 24 h: control 1.04±0.01 vs. stretch+losartan 1.13±0.1, p=0.69; 48 h: control 1.18±0.02 vs. stretch +losartan 1.12± 0.08, p < 0.63; n = 8 preparations each) (Fig. 6C). However, NT-3 expression of stretched neurocytes treated with losartan remained unaffected (NT-3: 0 h: control 1±0 vs. stretch+losartan 1±0; 24 h: control 0.97±0.01 vs. stretch+losartan 0.97±0.02, p=0.97; 48 h: control 0.94±0.01 vs. stretch 0.95±0.02, p=0.6; n=8 preparations each) (Fig. 6D). 3.7. Losartan prevents the stretch-induced growth response Next, we analyzed if losartan also prevents the stretch-induced increase of neurite outgrowth and GAP-43 protein expression. Losartan completely prevented the stretch-induced increase of neurite outgrowth (in µm: control 7.5 ± 1.7, stretch 25.9 ± 1.3, stretch + losartan 8.6 ± 1.6, n = 3 preparations each) (Fig. 7A). Furthermore, losartan abolished the stretch-induced increase of GAP-43 protein expression (control 1 ± 0, stretch 1.73 ± 0.21, stretch+ losartan 1.07 ± 0.06, n = 3 preparations each) (Fig. 7B). These results demonstrate that inhibition of the AT-1 receptors with losartan is a potential tool to prevent the stretch-induced growth of sympathetic neurocytes. Hereby, AT-1 receptor blockers possibly prevent the stretch-induced sympathetic hyperinnervation. However, further in-vivo studies are necessary to address this important issue. 4. Discussion This study demonstrates: i) cellular stretch leads to an increased growth of rat sympathetic neurocytes; ii) neonatal rat SCG neurocytes
express NGF, CNTF, NT-3, GDNF, but not BDNF mRNA and protein; iii) the stretch response is coupled to an increase of NGF and CNTF, while NT-3 remains unaffected, GDNF is down-regulated and BDNF could not be detected; iv) by specific neurotrophin neutralizing experiments, NGF and CNTF were identified as important stretch-induced neural growthinducing factors; v) AT-1 receptor inhibition with losartan prevents the stretch-induced alterations of neurotrophins; vi) losartan abolished the stretch-induced increase of neural growth in terms of preventing the stretch-induced increase of neurite outgrowth and GAP-43 protein expression. SNS is a major factor for the development of life-threatening arrhythmias after MI (Zhou et al., 1995) and congestive HF (Himura et al., 1993). Pathological outgrowth of sympathetic fibers and subsequent hyperinnervation seems to be an important contributor to an elevated sympathetic tone and has been reported to occur within the first hours of MI (Oh et al., 2006). There is evidence that inflammatory cells like myofibroblasts and macrophages contribute to the development of SNS in the ischemic injury area after MI (Hasan et al., 2006). If other contributing factors than inflammatory cells or NGF are involved in the pathogenesis of SNS, still needs to be investigated. Mechanical stretch is an ubiquitous pathophysiological stimulus accompanying several cardiac diseases like MI, systolic and diastolic HF or arterial hypertension (Zimmerman et al., 2000; Ashikaga et al., 2005; Gao et al., 2007; Koba et al., 2008). Recently, we could demonstrate that mechanical stretch of ventricular cardiomyocytes induces a significant down-regulation of NGF (Rana et al., 2009). However, a stretch-induced NGF down-regulation in cardiomyocytes would not explain the induction of SNS after MI and HF. To our knowledge, the impact of mechanical stretch on sympathetic neurocytes has yet not been investigated so far. In this study, we provide first evidence that mechanical stretch itself is able to induce
Fig. 6. Losartan prevents the stretch-induced alterations of neurotrophins. Neurocytes were treated with losartan (1 × 10− 6 mol/l) during the exposal of mechanical stretch. (A,D) Losartan completely prevented the stretch-induced up-regulation of NGF and CNTF in the cell culture supernatant (n = 8 preparations each). (C) Losartan abolished the stretch-induced downregulation of GDNF (n = 8 preparations each). (D) NT-3 expression of stretched neurocytes treated with losartan remained unaffected (n = 8 preparations each).
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Fig. 7. Losartan prevents the stretch-induced growth response. (A) Losartan completely prevented the stretch-induced increase of neurite outgrowth (n = 3 preparations each, # p < 0.05 vs. control, *p < 0.05 vs. stretch). (B) Losartan abolished the stretch-induced increase of GAP-43 protein expression (n = 3 preparations each, #p < 0.05 vs. control, *p < 0.05 vs. stretch).
growth of rat sympathetic neurocytes by enhancing the expression of NGF and CNTF in an autocrine/paracrine manner. The interactions of sympathetic neurons and their target tissues are not well understood, although the survival functions of targetderived neurotrophins such as NGF and NT-3 have been wellcharacterized. In the periphery, NGF, NT-3, CNTF and GDNF are produced by target tissues, internalized by the innervating sympathetic and/or sensory neuron, and retrogradely transported to the cell body (Zhou et al., 1997; Kuruvilla et al., 2004), where they carry out their neurotrophic activities. However, contradictive data exist about the intrinsic expression of NGF in neonatal rat SCG neurocytes. While previous experiments have failed to detect NGF production in cultured neonatal rat SCG (Kannan et al., 1996), Hasan et al. demonstrated that neonatal rat SCG neurocytes express NGF mRNA, which is maintained even when they are undergoing apoptosis (Hasan et al., 2003). Besides NGF, we also demonstrated that neonatal rat SCG neurocytes express NT-3, CNTF and GDNF mRNA and protein. However, BDNF gene and protein expression was not detectable in neonatal rat SCG neurocytes. These findings suggest that a potential neurotrophin-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system. To our knowledge, no data exists about the endogenous expression of NT-3, CNTF, GDNF or BDNF in neonatal rat SCG neurocytes.
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Blocking activation of the renin–angiotensin system (RAS) by inhibition of the angiotensin converting enzyme (ACE) or AT-1 receptor blockade after MI is a standard treatment to reverse maladaptive left ventricular remodeling (Milliez et al., 2009). Many studies have demonstrated that ACE inhibitors have beneficial effects on MI and congestive HF (Pfeffer et al., 1992; Hilfiker-Kleiner and Drexler, 2002). Blockade of the RAS with AT-1 antagonists has also been reported to have beneficial effects on MI and congestive HF (Pitt et al., 1997). On a molecular basis, it has been shown that angiotensin-II exposure to cultured fibroblasts promotes increased collagen synthesis (Brilla et al., 1994). Furthermore, activation of the RAS leads to cardiac fibrosis in a variety of pathological conditions. In experimental hypertension, angiotensin-II causes ventricular fibrosis that can be dissociated from hemodynamic load (Brilla et al., 1990). Moreover, inhibition of the ACE prevents collagen accumulation in the non-infarcted myocardium after acute myocardial infarction in rats (van Krimpen et al., 1991). In cardiac myocytes, angiotensin-II induces myocyte hypertrophy via the calcineurin-NFAT pathway, which can be abolished by AT-1 receptor blockade (Zobel et al., 2007). AT-1 receptor blockade has also been demonstrated to prevent stretch-induced electrical remodeling in neonatal rat atrial cardiomyocytes (Saygili et al., 2007). On neuronal basis, it has been demonstrated that neurons of the spinal cord (Ahmad et al., 2003) and the SCG (Tang et al., 2008) express the AT-1 receptor. In addition, Tang et al. demonstrated that most of the angiotensin-II receptors in the SCG were of the AT-1 type, while AT-2 receptors were scarce in the samples under study (Tang et al., 2008). Moreover, Patil et al. demonstrated that neurons of sympathetic coeliac ganglia express endogenous angiotensinogen and ACE (Patil et al., 2008). These studies indicate the presence of an intrinsic renin–angiotensin system in SCGs and one may hypothesize a functionally significant role for angiotensinII in this sympathetic system. However, if SCG neurocytes themselves synthesize, store and excrete angiotensin-II or if there is another, yet to be identified, endogenous ligand for the AT-1 receptor expressed in the culture still needs to be elucidated. We here provide first evidence that, besides structural, contractile and electrical remodeling, AT-1 blockade also affects neural remodeling by inhibition of the stretch-induced growth of sympathetic neurocytes. The stretch-induced inhibition of neural growth by losartan is coupled to the inhibition of the stretchinduced increase of the neurotrophins NGF and CNTF. How the AT-1 receptor mediates the effect on neurotrophin regulation still needs to be investigated. There are some limitations of the study which have to be addressed: i) besides cyclic stretch, static stretch addresses only one of the 2 stretch components to which intracardiac cells are exposed. ii) The model chosen herein employs neonatal rat sympathetic neurocytes of the SCG. In fact, very few SCG neurons (∼less than 5 %) project to the heart (Pardini et al., 1989; Richardson et al., 2006). SCG cells primarily project to targets within the head and neck (Flett and Bell, 1991). The stellate-middle cervical ganglion complex contains the largest percentage of cardiac-projecting sympathetic neurons among the thoracic ganglia (>90%) (Yang et al., 2006). However, because SCG neurocyte cell cultures constitute a wellcharacterized and relatively homogeneous population, we chose to investigate neurocytes of SCG as a model of sympathetic neurocytes. Furthermore, the genetic expression pattern of the investigated proteins may differ substantially between these cells and adult neurocytes. Since stretch exposition of adult neurocytes is still challenging we were not able to present data on stretched adult neurocytes. iii) In this study, we did not identify intracellular transduction pathways by which stretch increases NGF and CNTF via the AT-1 receptor. Further investigations are required to address this issue. Acknowledgements We thank Esra Saygili for her helpful assistance. This work contains data from the doctoral thesis of Dorothee Hommes (University RWTH Aachen, Germany).
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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Mechanical stretch induces nerve sprouting in rat sympathetic neurocytes Obaida R. Rana a,⁎, Patrick Schauerte a, Dorothee Hommes a, Robert H.G. Schwinger b, Jörg W. Schröder a, Rainer Hoffmann a, Erol Saygili a a b
Department of Cardiology, RWTH Aachen University, Aachen, 52074, Germany Medical Clinic II, Klinikum Weiden, Weiden, 92637, Germany
a r t i c l e
i n f o
Article history: Received 10 October 2009 Received in revised form 30 December 2009 Accepted 5 January 2010 Keywords: Sympathetic nerve sprouting SCG Mechanical stretch Heart failure Myocardial infarction NGF CNTF
a b s t r a c t Sympathetic nerve sprouting (SNS) has been shown to occur after myocardial infarction (MI) and heart failure (HF) and is known to be responsible for the development of lethal arrhythmias. During MI or HF intracardiac cells are exposed to increased mechanical stretch. Molecular mechanisms which trigger sympathetic neural growth are largely unknown. Therefore, this study aimed to investigate the impact of mechanical stretch on rat neonatal sympathetic neurocytes of the superior cervical ganglion (SCG). Mechanical stretch resulted in an increased growth of sympathetic neurocytes. Furthermore, we could demonstrate that SCG neurocytes express nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3) and glial derived neurotrophic factor (GDNF) on mRNA and protein level. An increased NGF and CNTF expression, a down-regulated GDNF expression and an unchanged NT-3 expression were identified in the neurocyte cell culture supernatant of neurocytes exposed to mechanical stretch. However, neither brain derived neurotrophic factor (BDNF) mRNA and protein was expressed in SCG neurocytes, nor BDNF could be detected in the cell culture supernatant of SCG neurons. By anti-neurotrophin neutralizing experiments NGF and CNTF were identified as important stretch-induced growth-inducing factors. Losartan, an angiotensin-II type 1 receptor inhibitor, abolished the stretch-induced increase of NGF and CNTF expression and thereby prevented the stretch-induced neural growth. This study provides new molecular mechanisms by which the inhibitory effect of angiotensin-II type 1 receptor blockers on the neural/arrhythmogenic remodeling can be explained. However, further in-vivo studies are required to address this important issue. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Sympathetic nerve sprouting (SNS) is a major factor for the development of malignant arrhythmias after myocardial infarction (MI) (Zhou et al., 1995) and congestive heart failure (HF) (Himura et al., 1993). Pathological outgrowth of sympathetic fibers and subsequent hyperinnervation seems to be an important contributor to an elevated sympathetic tone and has been reported to occur within the first hours of MI (Oh et al., 2006). Furthermore, SNS has been suggested to be a major contributor to sudden cardiac death (Cao et al., 2000a; Cao et al., 2000b). After MI increased nerve growth factor (NGF) expression has been shown to promote sympathetic hyperinnervation (Hasan et al., 2006), and may represent an adaptive mechanism by which the heart tries to maintain ventricular contractility at the cost of eventually triggered ventricular arrhythmias. There is evidence that inflammatory cells like myofibroblasts and macrophages contribute to the development of SNS in the ischemic injury area after MI (Hasan et al., 2006). If ⁎ Corresponding author. Department of Cardiology, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany. Tel.: +49 241 8035142; fax: +49 241 8082482. E-mail address: [email protected] (O.R. Rana). 1566-0702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2010.01.003
other contributing factors than inflammatory cells or NGF are involved in the pathogenesis of SNS still needs to be investigated. Mechanical stretch is such condition to which the cardiac cells are exposed after MI and during HF (Zimmerman et al., 2000; Ashikaga et al., 2005; Gao et al., 2007; Koba et al., 2008). Recently, we could demonstrate that mechanical stretch of ventricular cardiomyocytes induces a significant down-regulation of NGF (Rana et al., 2009). However, a stretch-induced NGF down-regulation in cardiomyocytes would not explain the induction of SNS after MI and HF. Because the myocardium in extensively innervated by the sympathetic nervous system and the impact of mechanical stretch on sympathetic neurocytes is unknown, this study aimed to investigate the impact of mechanical stretch on rat neonatal sympathetic neurocytes of the superior cervical ganglion (SCG). Furthermore, stretch-induced alterations of the neurotrophins NGF, neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), glial derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) were investigated. Mechanical stretch resulted in increased neural growth assessed by measuring the neurite outgrowth and growth associated protein 43 (GAP-43) expression. Furthermore, in the cell culture supernatant of neurocytes exposed to mechanical stretch NGF and CNTF expression was significantly increased, GDNF expression was down-regulated, NT-3 expression was unchanged,
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while BDNF could not be detected. By anti-neurotrophin neutralizing experiments, NGF and CNTF were identified as important stretchinduced neural growth-inducing factors. Angiotensin-II type 1 (AT-1) receptor blockade with losartan completely prevented the stretchinduced neural growth by attenuating the stretch-induced increase of NGF and CNTF. 2. Materials and methods 2.1. Superior cervical ganglia (SCG) neurocyte cell culture All animals were cared for according to institutional animal-care requirements. Primary cultures of SCGs were performed from postnatal day 1 to 3 Sprague Dawley rats (Charles River, Germany) as described previously (Rana et al., 2009). Briefly, both SCG of 20–30 neonatal rat were dissected from the carotid bifurcation, cut into small pieces, and then transferred into 1 ml of Neuronal Base Medium (PAA, U15-024) supplemented with collagenase II (1.0 mg/ml, 280 U/ mg, Biochrom, C2-22), trypsin (0.5 mg/ml, trypsin 1:250, PAA, L-11-
002) and 1% penicillin–streptomycin (Sigma, P4458). Enzymatic digestion was performed for 40 min at 37 °C. Thereafter, SCGs were dissociated mechanically with fire-polished glass pipettes and centrifuged at 3000 rpm for 2 min. Cells were resuspended in 5 ml Neuronal Base Medium supplemented with 10% fetal bovine serum (FBS, PAA, A-15-101) and 1% penicillin–streptomycin. Cells were incubated for 2 h in an incubator (95% air, 5% CO2, 100% humidity) to allow non-neuronal cell (e.g. fibrocytes and Schwann cells) to attach, while neuronal cells need longer incubation duration to attach. After 2 h of incubation cell supernatant was centrifuged at 3000 rpm for 2 min and cells were resuspended in final Neuronal Base Medium supplemented with 10% FBS, 1% penicillin–streptomycin, 2 mM Lalanyl–L-glutamine (Gibco Invitrogen Corporation) and 10 µM 5Bromo-2′-deoxy-uridine (5-BrdU, Sigma, B-9285). Cells were plated onto silicone membranes coated with collagen type I (Bioflex Collagen I 6-well plates, Dunn, BF-3001C) at a density of 100,000 cells per well. With this procedure we could achieve purity of 93% of tyrosine hydroxylase (TH) positive neuronal cells (3 cell preparations, 635 cells of total 684 cells were TH positive by immunofluorescence, data
Fig. 1. Mechanical stretch induces growth of rat neonatal sympathetic neurocytes. (A) Measurements of neurite outgrowth in µm, n = 3 preparations each, *p < 0.01 vs. stretch 0 day, (B) net increase of neurite outgrowth in µm, n = 3 preparations each, *p < 0.01 vs. stretch, (C) representative and quantitation of GAP-43 Western blots, n = 3 preparations, *p < 0.01 vs. stretch, (D) representative images of control and stretched neurocytes in cell culture, scale bar 20 µm, and (E) representative tyrosine hydroxylase (TH) and growth associated protein 43 (GAP-43) immunofluorescence images of stretched neurocytes demonstrating the sympathetic origin of the cultivated neurocytes.
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(steps: 3, 7, 13 %) (Rana et al., 2009). Un-stretched control neurocytes were treated equally without application of mechanical stretch. 2.3. Antibodies and neurotrophin neutralizing antibodies
Fig. 2. mRNA expression of NGF, CNTF, NT-3 and GDNF in neonatal rat SCG neurocytes. Real-time PCR experiments of SCG neurocytes were performed after 72 h of stretch application. Stretch resulted in a significant up-regulation of NGF and CNTF mRNA (n = 3 preparations each). The mRNA expression of NT-3 was unchanged in stretched neurocytes compared to un-stretched control neurocytes (n = 3 preparations each). The gene expression of GDNF was significantly down-regulated in stretched neurocytes (n = 3 preparations each). The BDNF gene expression in control and stretched SCG neurocytes was not detectable, *p < 0.02 vs. control.
not shown). During the first 48 h SCG neurocytes were grown in serum-containing medium. Thereafter, stretch was introduced to attached neurocytes by applying a gradual increase of tension over a period of 72 h in a complete serum-free condition.
2.2. Application of homogeneous equibiaxial static stretch Static stretch was introduced to attached neurocytes by applying a gradual increase of tension as described previously (Rana et al., 2008). Because myocardium dilatation in most cases is a chronic adaptive response of cells to mechanical load neurocytes were exposed to a gradual increase of stretch every 24 h from 3 to 13% over 3 days
Following primary antibodies were used: rabbit anti-NGF (Santa Cruz, sc-548), rabbit anti-NT-3 (Santa Cruz, sc-547), rabbit anti-BDNF (Santa Cruz, sc-546), goat anti-CNTF (Santa Cruz, sc-1912), rabbit anti-GDNF (Santa Cruz, sc-328), rabbit anti-GAP-43 (Santa Cruz, sc10786), rabbit anti-tyrosine hydroxylase (Santa Cruz, sc-14007) and rabbit anti-GAPDH (Cell Signaling, #2118). Neurotrophin neutralizing antibodies: anti-NGF (Sigma-Aldrich, #N6655, 1:500 dilution), anti-CNTF (R&D Systems, #MAB557, 5 µg/ml), anti-GDNF (R&D Systems, #MAB212, 5 µg/ml) and anti-NT-3 (Promega, #G1651, 5 µg/ml). 2.4. RNA preparation, first-strand cDNA synthesis and quantitative realtime reverse transcription-PCR RNA extraction, first-strand cDNA synthesis and quantitative real-time PCR experiments were performed as described previously (Rana et al., 2008). Each preparation contained sufficient mRNA concentration for first-strand cDNA synthesis. PCR primers and fluorogenic probes for NGF, CNTF, GDNF, NT-3, BDNF and the endogenous control were purchased from Applied Biosystems (Foster City, CA). The assay numbers were as follows: Rn01533872_m1 (Ngfb), Rn00755092_m1 (Cntf), Rn00569510_m1 (Gdnf), Rn00579280_m1 (Ntf3), Rn00560868_m1 (Bdnf), and Rn00560865_m1 (beta-2 microglobulin). 2.5. Neurite outgrowth To measure the net increase of neurite outgrowth of SCG neurocytes, 10 light-optical microscopic images (200× magnification) were taken
Fig. 3. Neonatal rat SCG neurocytes express NGF, CNTF, NT-3 and GDNF. Representative immunofluorescence images of NGF, CNTF, NT-3 and GDNF in cultured neonatal rat SCG neurocytes. No BDNF-immunostaining could be revealed in cultured neonatal rat SCG neurocytes (image not shown).
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before (0 h) and after (72 h) the application of mechanical stretch of each preparation. The mean length of all neurocyte cell extensions was measured with the help of a scale bar on each microscopic image. The net increase of neurite outgrowth was assessed by subtraction of the mean neurite outgrowth after 72 h of stretch from the mean neurite outgrowth before stretch application. For each condition at least 3 cell preparation were performed with 1 well (100,000 cells). Number of neurocytes considered for neurite outgrowth measurement before/after stretch application: 641(before)/518(after) control cells (10 preparations), 603/479 stretched cells (10 preparations), 181/137 stretched cells + anti-NGF (3 preparations), 170/143 stretched cells + anti-NT-3 (3 preparations), 176/155 stretched cells + anti-CNTF (3 preparations), 199/135 stretched cells+ anti-GDNF (3 preparations) and 197/147 stretched cells + losartan (3 preparations). 2.6. ELISA, Western blot and immunofluorescence At 0, 24 and 72 h, the conditioned medium was collected and assayed. NGF, CNTF, GDNF, NT-3 and BDNF were assessed by ELISA kits from R&D Systems (Minneapolis, USA) according to the manufacturer's protocol. Western blot and immunofluorescence experiments were performed as described previously (Rana et al., 2009; Saygili et al., 2009). Cell lysis of each SCG preparation contained sufficient protein concentration to perform Western blot experiments for GAP-43 and GAPDH detection.
Figs. 3 and 5 were analyzed by 2-way-ANOVA followed by Bonferroni's post-hoc test. p-values <0.05 were considered as statistically significant. 3. Results 3.1. Mechanical stretch induces growth of neonatal rat sympathetic neurocytes Mechanical stretch of SCG neurocytes over a time period of 72 h resulted in a significant increase of neurite outgrowth compared to control neurocytes (in µm: control: 0 h = 41.9 ± 1.3 vs. 72 h = 49.4 ±2.4, p=0.13, n=3 preparations; stretch: 0 h=45.1±0.9 vs. 72 h=71± 1.7, p<0.01, n=3 preparations) (Fig. 1A). The net increase of neurite outgrowth of stretched neurocytes after 72 h was significantly increased compared to un-stretched control neurocytes (control: 7.5±1.7 vs. stretch: 25.9±1.3, n=3 preparations, p<0.01) (Fig. 1B). Furthermore, the GAP-43 protein expression, a marker for neural growth, was significantly increased in stretched neurocytes compared to un-stretched control neurocytes (control: 1±0 vs. stretch: 1.35±0.12, n=3 preparations each, p<0.01) (Fig. 1C). Fig. 1D demonstrates representative images of control and stretched neurocytes in cell culture. To demonstrate the sympathetic origin of our cultivated neurocytes, tyrosine hydroxylase (TH) and GAP-43 immunofluorescence experiments were performed on stretched neurocytes (Fig. 1E). 3.2. Neonatal rat SCG neurocytes express NGF, CNTF, NT-3 and GDNF mRNA
2.7. Statistical analysis All values are expressed as mean±SEM. Comparisons of two groups were performed by Student's t-test and multiple groups presented in
Next, we performed real-time PCR experiments of SCG neurocytes after 72 h of stretch application. The expression of the neurotrophins NGF, NT-3, GDNF, CNTF and BDNF was analyzed (Fig. 2). Stretch resulted in a
Fig. 4. Mechanical stretch results in up-regulation of NGF and CNTF and down-regulation of GDNF, while NT-3 demonstrates an unchanged protein expression pattern. (A,B) Mechanical stretch resulted in a significant up-regulation of NGF and CNTF concentration in the cell culture supernatant of stretched sympathetic neurocytes compared to un-stretched control neurocytes after 24 and 72 h assessed by ELISA. #p < 0.01 vs. control 24 h, *p < 0.01 vs. control 72 h. (C) The expression of GDNF was significantly down-regulated in stretched neurocytes after 72 h. *p < 0.01 vs. control 72 h (D) Mechanical stretch showed no significant effect on the NT-3 expression compared to un-stretched control neurocytes.
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significant up-regulation of NGF and CNTF mRNA in stretched neurocytes (NGF: control 1±0 vs. stretch 1.2±0.01, n=3 preparations each, p<0.02; CNTF: control 1±0 vs. stretch 1.43±0.03, n=3 preparations each, p < 0.02). The mRNA expression of NT-3 was unchanged in stretched neurocytes compared to un-stretched control neurocytes (NT-3: control 1 ± 0 vs. stretch 0.96 ± 0.05, n = 3 preparations each, p = 0.65). Furthermore, the gene expression of GDNF was significantly down-regulated in stretched neurocytes (GDNF: control 1 ± 0 vs. stretch 0.78 ± 0.01, n = 3 preparations each, p < 0.01). However, the BDNF gene expression in control and stretched SCG neurocytes was not detectable. 3.3. Detection of NGF, CNTF, NT-3 and GDNF protein expression in neonatal rat SCG neurocytes by immunofluorescence To investigate if neonatal rat SCG neurocytes express neurotrophins on protein level, NGF, CNTF, NT-3, GDNF and BDNF were analyzed by immunofluorescence in un-stimulated neurocytes. The purity of our cell culture was 93% as described in the Materials and methods section. By immunofluorescence experiments we could demonstrate that SCG neurocytes express NGF, CNTF, NT-3 and GDNF (Fig. 3). However, BDNF protein expression could not be revealed (image not shown).
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tions each) (Fig. 5A). In analogy to the neurite outgrowth measurements, stretch resulted in a significant increase in GAP-43 protein expression. NT-3 or GDNF neutralizing antibodies showed no effect on the stretch-induced increase in GAP-43 protein expression. However, NGF or CNTF neutralizing antibodies completely abolished the stretchinduced increase of GAP-43 protein expression (control 1 ± 0, stretch 1.6 ± 0.1, anti-NT3 1.6 ± 0.1, anti-GDNF 1.7 ± 0.1, anti-NGF 1 ± 0.1, antiCNTF 1.1 ± 0.1, n = 3 preparations each) (Fig. 5B). 3.6. Losartan prevents the stretch-induced alterations of neurotrophins In order to investigate the impact of AT-1 receptor inhibition on the stretch-induced growth response, neurocytes were treated with losartan (1×10− 6 mol/l) during mechanical stretch. Losartan completely prevented the stretch-induced up-regulation of NGF and CNTF in the cell culture supernatant (NGF: 0 h: control 1±0 vs. stretch+losartan 1±0; 24 h: control 1.02±0.01 vs. stretch+losartan 1.02±0.01, p=0.34; 48 h: control 1.1 ± 0.01 vs. stretch+ losartan 1.13± 0.03, p = 0.93; n = 8 preparations each) (CNTF: 0 h: control 1±0 vs. stretch+losartan 1±0; 24 h: control 0.99±0.01 vs. stretch+losartan 0.97±0.01, p=0.29; 48 h: control 1.02±0.01 vs. stretch+losartan 0.96±0.01, p=0.17; n=8
3.4. Mechanical stretch results in up-regulation of NGF and CNTF and down-regulation of GDNF, while NT-3 demonstrates an unchanged protein expression pattern Mechanical stretch resulted in a significant up-regulation of NGF and CNTF concentration in the cell culture supernatant of stretched sympathetic neurocytes compared to un-stretched control neurocytes after 24 and 72 h assessed by ELISA (NGF: 0 h: control 1±0 vs. stretch 1± 0; 24 h: control 1.02±0.01 vs. stretch 1.16±0.01, p<0.01; 48 h: control 1.1±0.01 vs. stretch 1.35±0.01, p<0.01; n=8 preparations each) (CNTF: 0 h: control 1±0 vs. stretch 1±0; 24 h: control 0.99±0.01 vs. stretch 1.2±0.01, p<0.01; 48 h: control 1.02±0.01 vs. stretch 1.41± 0.01, p<0.01; n=8 preparations each) (Fig. 4A and B). The expression of GDNF was significantly down-regulated in stretched neurocytes after 72 h (GDNF: 0 h: control 1±0 vs. stretch 1±0; 24 h: control 1.04±0.01 vs. stretch 0.83±0.02, p=0.07; 48 h: control 1.18±0.02 vs. stretch 0.69± 0.03, p<0.01; n=8 preparations each) (Fig. 4C). However, mechanical stretch demonstrated no significant effect on NT-3 expression as compared to un-stretched control neurocytes (NT-3: 0 h: control 1±0 vs. stretch 1±0; 24 h: control 0.97±0.01 vs. stretch 0.96±0.01, p=0.82; 48 h: control 0.94±0.01 vs. stretch 0.9±0.01, p=0.29; n=8 preparations each) (Fig. 4D). In analogy to the gene expression results, the neurocyte cell culture supernatant was free of BDNF. By 2-way-ANOVA analysis the means of values for 24 h and 72 h of NGF and CNTF demonstrated a statistical significance (p<0.05) compared to the means of 0 h. Furthermore, the means of 24 h were statistical significant as compared to 72 h for NGF and CNTF. GDNF and NT-3 demonstrated nonsignificant mean values as compared to all 3 time points. 3.5. NGF and CNTF are important stretch-induced sympathetic neurocyte growth-inducing neurotrophins To investigate which neurotrophic factors are involved in the stretchinduced neural growth of rat sympathetic neurocytes, stretched neurocytes were co-treated with anti-neurotrophin neutralizing antibodies and the net increase of neurite outgrowth (Fig. 5A) and GAP-43 protein expression (Fig. 5B) experiments were performed. NGF or CNTF neutralizing antibodies abolished the stretch-induced increase of neurite outgrowth, while NT-3 or GDNF neutralizing antibodies had no influence on the stretch-induced increase of neurite outgrowth (in µm: control 7.5 ± 1,7, stretch 25.9± 1.3, anti-NT-3 24.2 ± 1.0, antiGDNF 27.6 ± 1.4, anti-NGF 6 ± 2.8, anti-CNTF 13.7 ± 0.4, n = 3 prepara-
Fig. 5. NGF and CNTF are important stretch-induced sympathetic neurocyte growthinducing neurotrophins. (A) Stretched neurocytes were co-treated with anti-neurotrophin neutralizing antibodies and the net increase of neurite outgrowth was assessed. NGF or CNTF neutralizing antibodies abolished the stretch-induced increase of neurite outgrowth, while NT-3 or GDNF neutralizing antibodies had no influence on the stretch-induced increase of neurite outgrowth (n = 3 preparations each, #p < 0.05 vs. control, *p < 0.05 vs. stretch). (B) Stretch resulted in a significant increase in GAP-43 protein expression. NT-3 or GDNF neutralizing antibodies showed no effect on the stretch-induced increase in GAP-43 protein expression. NGF or CNTF neutralizing antibodies completely abolished the stretch-induced increase of GAP-43 protein expression (n = 3 preparations each, #p < 0.05 vs. control, *p < 0.05 vs. stretch).
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preparations each) (Fig. 6A and B). Furthermore, losartan abolished the stretch-induced down-regulation of GDNF (GDNF: 0 h: control 1±0 vs. stretch+losartan 1±0; 24 h: control 1.04±0.01 vs. stretch+losartan 1.13±0.1, p=0.69; 48 h: control 1.18±0.02 vs. stretch +losartan 1.12± 0.08, p < 0.63; n = 8 preparations each) (Fig. 6C). However, NT-3 expression of stretched neurocytes treated with losartan remained unaffected (NT-3: 0 h: control 1±0 vs. stretch+losartan 1±0; 24 h: control 0.97±0.01 vs. stretch+losartan 0.97±0.02, p=0.97; 48 h: control 0.94±0.01 vs. stretch 0.95±0.02, p=0.6; n=8 preparations each) (Fig. 6D). 3.7. Losartan prevents the stretch-induced growth response Next, we analyzed if losartan also prevents the stretch-induced increase of neurite outgrowth and GAP-43 protein expression. Losartan completely prevented the stretch-induced increase of neurite outgrowth (in µm: control 7.5 ± 1.7, stretch 25.9 ± 1.3, stretch + losartan 8.6 ± 1.6, n = 3 preparations each) (Fig. 7A). Furthermore, losartan abolished the stretch-induced increase of GAP-43 protein expression (control 1 ± 0, stretch 1.73 ± 0.21, stretch+ losartan 1.07 ± 0.06, n = 3 preparations each) (Fig. 7B). These results demonstrate that inhibition of the AT-1 receptors with losartan is a potential tool to prevent the stretch-induced growth of sympathetic neurocytes. Hereby, AT-1 receptor blockers possibly prevent the stretch-induced sympathetic hyperinnervation. However, further in-vivo studies are necessary to address this important issue. 4. Discussion This study demonstrates: i) cellular stretch leads to an increased growth of rat sympathetic neurocytes; ii) neonatal rat SCG neurocytes
express NGF, CNTF, NT-3, GDNF, but not BDNF mRNA and protein; iii) the stretch response is coupled to an increase of NGF and CNTF, while NT-3 remains unaffected, GDNF is down-regulated and BDNF could not be detected; iv) by specific neurotrophin neutralizing experiments, NGF and CNTF were identified as important stretch-induced neural growthinducing factors; v) AT-1 receptor inhibition with losartan prevents the stretch-induced alterations of neurotrophins; vi) losartan abolished the stretch-induced increase of neural growth in terms of preventing the stretch-induced increase of neurite outgrowth and GAP-43 protein expression. SNS is a major factor for the development of life-threatening arrhythmias after MI (Zhou et al., 1995) and congestive HF (Himura et al., 1993). Pathological outgrowth of sympathetic fibers and subsequent hyperinnervation seems to be an important contributor to an elevated sympathetic tone and has been reported to occur within the first hours of MI (Oh et al., 2006). There is evidence that inflammatory cells like myofibroblasts and macrophages contribute to the development of SNS in the ischemic injury area after MI (Hasan et al., 2006). If other contributing factors than inflammatory cells or NGF are involved in the pathogenesis of SNS, still needs to be investigated. Mechanical stretch is an ubiquitous pathophysiological stimulus accompanying several cardiac diseases like MI, systolic and diastolic HF or arterial hypertension (Zimmerman et al., 2000; Ashikaga et al., 2005; Gao et al., 2007; Koba et al., 2008). Recently, we could demonstrate that mechanical stretch of ventricular cardiomyocytes induces a significant down-regulation of NGF (Rana et al., 2009). However, a stretch-induced NGF down-regulation in cardiomyocytes would not explain the induction of SNS after MI and HF. To our knowledge, the impact of mechanical stretch on sympathetic neurocytes has yet not been investigated so far. In this study, we provide first evidence that mechanical stretch itself is able to induce
Fig. 6. Losartan prevents the stretch-induced alterations of neurotrophins. Neurocytes were treated with losartan (1 × 10− 6 mol/l) during the exposal of mechanical stretch. (A,D) Losartan completely prevented the stretch-induced up-regulation of NGF and CNTF in the cell culture supernatant (n = 8 preparations each). (C) Losartan abolished the stretch-induced downregulation of GDNF (n = 8 preparations each). (D) NT-3 expression of stretched neurocytes treated with losartan remained unaffected (n = 8 preparations each).
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Fig. 7. Losartan prevents the stretch-induced growth response. (A) Losartan completely prevented the stretch-induced increase of neurite outgrowth (n = 3 preparations each, # p < 0.05 vs. control, *p < 0.05 vs. stretch). (B) Losartan abolished the stretch-induced increase of GAP-43 protein expression (n = 3 preparations each, #p < 0.05 vs. control, *p < 0.05 vs. stretch).
growth of rat sympathetic neurocytes by enhancing the expression of NGF and CNTF in an autocrine/paracrine manner. The interactions of sympathetic neurons and their target tissues are not well understood, although the survival functions of targetderived neurotrophins such as NGF and NT-3 have been wellcharacterized. In the periphery, NGF, NT-3, CNTF and GDNF are produced by target tissues, internalized by the innervating sympathetic and/or sensory neuron, and retrogradely transported to the cell body (Zhou et al., 1997; Kuruvilla et al., 2004), where they carry out their neurotrophic activities. However, contradictive data exist about the intrinsic expression of NGF in neonatal rat SCG neurocytes. While previous experiments have failed to detect NGF production in cultured neonatal rat SCG (Kannan et al., 1996), Hasan et al. demonstrated that neonatal rat SCG neurocytes express NGF mRNA, which is maintained even when they are undergoing apoptosis (Hasan et al., 2003). Besides NGF, we also demonstrated that neonatal rat SCG neurocytes express NT-3, CNTF and GDNF mRNA and protein. However, BDNF gene and protein expression was not detectable in neonatal rat SCG neurocytes. These findings suggest that a potential neurotrophin-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system. To our knowledge, no data exists about the endogenous expression of NT-3, CNTF, GDNF or BDNF in neonatal rat SCG neurocytes.
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Blocking activation of the renin–angiotensin system (RAS) by inhibition of the angiotensin converting enzyme (ACE) or AT-1 receptor blockade after MI is a standard treatment to reverse maladaptive left ventricular remodeling (Milliez et al., 2009). Many studies have demonstrated that ACE inhibitors have beneficial effects on MI and congestive HF (Pfeffer et al., 1992; Hilfiker-Kleiner and Drexler, 2002). Blockade of the RAS with AT-1 antagonists has also been reported to have beneficial effects on MI and congestive HF (Pitt et al., 1997). On a molecular basis, it has been shown that angiotensin-II exposure to cultured fibroblasts promotes increased collagen synthesis (Brilla et al., 1994). Furthermore, activation of the RAS leads to cardiac fibrosis in a variety of pathological conditions. In experimental hypertension, angiotensin-II causes ventricular fibrosis that can be dissociated from hemodynamic load (Brilla et al., 1990). Moreover, inhibition of the ACE prevents collagen accumulation in the non-infarcted myocardium after acute myocardial infarction in rats (van Krimpen et al., 1991). In cardiac myocytes, angiotensin-II induces myocyte hypertrophy via the calcineurin-NFAT pathway, which can be abolished by AT-1 receptor blockade (Zobel et al., 2007). AT-1 receptor blockade has also been demonstrated to prevent stretch-induced electrical remodeling in neonatal rat atrial cardiomyocytes (Saygili et al., 2007). On neuronal basis, it has been demonstrated that neurons of the spinal cord (Ahmad et al., 2003) and the SCG (Tang et al., 2008) express the AT-1 receptor. In addition, Tang et al. demonstrated that most of the angiotensin-II receptors in the SCG were of the AT-1 type, while AT-2 receptors were scarce in the samples under study (Tang et al., 2008). Moreover, Patil et al. demonstrated that neurons of sympathetic coeliac ganglia express endogenous angiotensinogen and ACE (Patil et al., 2008). These studies indicate the presence of an intrinsic renin–angiotensin system in SCGs and one may hypothesize a functionally significant role for angiotensinII in this sympathetic system. However, if SCG neurocytes themselves synthesize, store and excrete angiotensin-II or if there is another, yet to be identified, endogenous ligand for the AT-1 receptor expressed in the culture still needs to be elucidated. We here provide first evidence that, besides structural, contractile and electrical remodeling, AT-1 blockade also affects neural remodeling by inhibition of the stretch-induced growth of sympathetic neurocytes. The stretch-induced inhibition of neural growth by losartan is coupled to the inhibition of the stretchinduced increase of the neurotrophins NGF and CNTF. How the AT-1 receptor mediates the effect on neurotrophin regulation still needs to be investigated. There are some limitations of the study which have to be addressed: i) besides cyclic stretch, static stretch addresses only one of the 2 stretch components to which intracardiac cells are exposed. ii) The model chosen herein employs neonatal rat sympathetic neurocytes of the SCG. In fact, very few SCG neurons (∼less than 5 %) project to the heart (Pardini et al., 1989; Richardson et al., 2006). SCG cells primarily project to targets within the head and neck (Flett and Bell, 1991). The stellate-middle cervical ganglion complex contains the largest percentage of cardiac-projecting sympathetic neurons among the thoracic ganglia (>90%) (Yang et al., 2006). However, because SCG neurocyte cell cultures constitute a wellcharacterized and relatively homogeneous population, we chose to investigate neurocytes of SCG as a model of sympathetic neurocytes. Furthermore, the genetic expression pattern of the investigated proteins may differ substantially between these cells and adult neurocytes. Since stretch exposition of adult neurocytes is still challenging we were not able to present data on stretched adult neurocytes. iii) In this study, we did not identify intracellular transduction pathways by which stretch increases NGF and CNTF via the AT-1 receptor. Further investigations are required to address this issue. Acknowledgements We thank Esra Saygili for her helpful assistance. This work contains data from the doctoral thesis of Dorothee Hommes (University RWTH Aachen, Germany).
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