Cardiac autonomic nerve fiber regeneration in chronic heart failure
Do Akt gene-transduced mesenchymal stem cells promote repair? *☆

Hongliang Kong1, Zhanquan Li2, Shumei Zhao3, Li Zhu3, Yingjun Zhao2, Weiwei Zhang2, Guiping Xu2, Wenjun Hao2, Huijun Li2, Guoxian Qi1

1Department of Cardiology, the First Affiliated Hospital of China Medical University, Shenyang  110001, Liaoning Province, China
2Department of Cardiology, the People’s Hospital of Liaoning Province, Shenyang  110016, Liaoning Province, China
3Shenyang Normal University, Shenyang  110034, Liaoning Province, China

Hongliang Kong☆, Doctor, Associate chief physician, Department of Cardiology, First Affiliated Hospital of China Medical University, Shenyang  110001, Liaoning Province, China

Corresponding author: Guoxian Qi, Doctor, Professor, Department of Cardiology, the First Affiliated Hospital of China Medical University, Shenyang   110001, Liaoning Province, China   qgx2002@medmail.com.cn

Supported by: Scientific Research Program of Higher Education Institute in Liaoning Province, No. 2008S248*

Abstract
BACKGROUND: Transplantation of Akt-over-expressing mesenchymal stem cells (Akt-MSCs) has been shown to repair infarcted myocardium and improve cardiac function. However, little is known about the therapeutic effects of Akt-MSCs on cardiac autonomic neuropathy in chronic heart failure (CHF).
OBJECTIVE: The present study used adriamycin-induced CHF rat models to observe the effect of Akt-MSCs on cardiac autonomic nervous regeneration and the factors mediating this effect.
DESIGN, TIME AND SETTING: A randomized, controlled animal experiment was performed at the Central Laboratory of Basic Medical College, China Medical University, between September 2008 and April 2009.
MATERIALS: Rabbit anti-choline acetyltransferase (ChAT), growth associated protein-43 (GAP-43), synaptophysin (SYN) polyclonal antibodies and the secondary antibody (goat anti-rabbit IgG) were purchased from Boster, China. Cat-A-Kit assay system was provided by Amersham, USA.
METHODS: (1) Adult rat MSCs were isolated and cultured for the preparation of Akt-MSCs. (2) Forty male Wistar rats were intramyocardially administered adriamycin at 2 mg/kg over 3 days for a total of five times and once a week for additional five times thereafter to establish CHF models. At 2 weeks after final adriamycin treatment, 34 successful CHF rat models were randomized to three groups: Akt-MSCs (n = 11), simple MSCs (s-MSCs, n =11), and control (n = 12). Each group was intravenously administered Akt-MSCs (2×106 cells in 100 μL PBS), s-MSCs (2×106 cells in 100 μL PBS) or equal volume of phosphate buffered saline, once a day for a total of three times.
MAIN OUTCOME MEASURES: At 4 weeks after final adriamycin treatment, myocardial norepi-nephrine (NE) content was detected using a Cat-A-Kit assay system. Myocardial ChAT, SYN and GAP-43 were performed by immunohistochemistry and Western blot analysis. Prior to, 2 and 4 weeks after adriamycin treatment, echocardiographic examination was performed and left ven-tricular ejection fraction (LVEF) was determined.
RESULTS: Myocardial NE content, as well as SYN-positive and GAP-43-positive nerve fiber density and expression, and LVEF, was the greatest in the Akt-MSCs group, followed by the s-MSCs group, and lastly the control group (P < 0.05 or P < 0.01). ChAT expression was similar between Akt-MSCs and s-MSCs groups, but it was higher compared with the control group (P < 0.05). NE contents were negatively correlated to LVEF (r = –0.64, P = 0.015).
CONCLUSION: Transplantation of MSCs, in particular Akt-MSCs, promotes cardiac nervous re-generation in failing heart, which might be mediated by GAP-43.
Key Words: mesenchymal stem cells; Akt gene transfection; chronic heart failure; neural regeneration; autonomic nerve system

INTRODUCTION
   
The progressive remodeling of the heart, involving a loss of cardiomyocytes and an increase in fibroblasts, is an important cause of chronic heart failure (CHF)[1]. In addition, the intrinsic cardiac nervous system, in par-ticular the sympathetic nervous system, also contributes to the development of CHF[2]. Mesenchymal stem cells (MSCs) are pluri-potent cells[3] that can differentiate into neurons, vascular endothelial cells, and cardiomyocytes ex vivo. Their effect im-proving cardiac function has shown promise in both experimental animal heart models and human studies[3-7], which may be related to the repair and regeneration of cardio-myocytes, proapoptotic factors and their paracrine effects[3-7]. MSCs are an excellent carrier of therapeutic genes. The transduc-tion of MSCs with the Akt gene, supports their survival and protects against hy-poxia-induced apoptosis[8-9]. Furthermore, Akt-over-expressing MSCs have been shown to repair infarcted myocardium and  
improves cardiac function, either directly or indirectly, by preventing the apoptosis of cell grafts, enhancing their survival and improving paracrine mechanisms[10-12]. It is well known that the over-activity of parasympathetic tone aggravates the development of CHF. However, little information is available about the therapeutic potential of Akt-MSCs and MSCs on cardiac autonomic nerve activ-ity in CHF, including whether they improve cardiac func-tion by adjusting cardiac autonomic nervous regenera-tion. Therefore, the purpose of this study was to investi-gate whether the transplantation of MSCs transduced with a retroviral vector expressing Akt1 (Akt-MSCs) can adjust sympathetic and cholinergic nerve activity and whether their beneficial effects are mediated by nerve fiber sprouting.  

MATERIALS AND METHODS

Design
A randomized, controlled animal experiment.
Time and setting
This study was performed at the Central Laboratory of Basic Medical College of China Medical University be-tween September 2008 and April 2009.
Materials
Forty male Wistar rats of clean grade, aged 3 months, weighing (250 ± 10) g, were purchased from Laboratory Animal Center of China Medical University (certificate No. SCXK (Liao) 2003-0009). Experiments were performed in accordance with the Institutional Guidelines for Care and Use of Laboratory Animals and approved by Animal Ethics Committee, China Medical University.
Main reagents and instruments used are listed as follows:

 
 
Methods
Cell culture
According to previously published reports[8-9], rat MSCs were isolated and cultured. In brief, bone marrow cells were centrifuged under a density gradient (Percoll gra-dient being 1.074 g/mL and 1.070 g/mL, respectively), washed twice for 10 minutes each with 10 mL phos-phate-buffered saline (PBS, pH 7.2–7.4), re-suspended in 10 mL L-DMEM containing 10% FBS, and cultured at 37 °C in a 5% CO2 atmosphere. Culture medium was renewed every 2–3 days to discard hematopoietic cells, fibroblasts and other non-adherent cells.
Retroviral transduction of MSCs
Following the manufacturer’s protocol, as described by Kong et al[9-10], both the therapeutic (Akt1-encoding) and control (empty) pLHCX retroviral vectors were con-structed. The PCR primers for the amplification of the Akt1 cDNA were as follows: forward primer 5′-CTA GTT AAG CTT ATG GGG AGC AGC-3′; reverse primer 5′-GAT ATG ATC GAT TGA TCA GAG GGT TTA -3′. Passage 2 MSCs (2 × 105) were transduced with a ret-roviral vector expressing Akt1 (Akt-MSCs) in 6-well plates.
Establishment of CHF models and animal grouping
In accordance with previous experiments[13-14], 40 male Wistar rats were intramyocardially administered adria-mycin at 2 mg/kg five times over 3 days, followed by an additional five times over 1 week. At 2 weeks after final administration, 34 successful CHF rat models[13-14] were randomized into three groups: Akt-MSCs (n = 11), simple MSCs (s-MSCs, i.e., without Akt gene transfected, n = 11), and control (i.e., the same volume of PBS, n = 12). Each group was intravenously administered Akt-MSCs (2 × 106 cells in 100 μL PBS), s-MSCs (2×106 cells in 100 μL PBS) or an equal volume of PBS (control), once a day for 3 days. All rats were fed routine food and sacrificed after echocardiography at 4 weeks.
Myocardial NE contents
Left ventricular tissues from the anterior muscle were rapidly removed to radioenzymatically measure tissue norepinephrine (NE) contents using a Cat-A-Kit assay system[15]. Tissue samples were homogenized using a Brinkman Polytron PCU-2 homogenizer (8-s burst × 3 at setting 8) and resuspended in 0.4 mmol/L perchloric acid (pH 7.4) with 5 mmol/L reduced glutathione. This was followed by centrifugation at 500 × g for 10 minutes, after which the supernatant was removed for the radioenzy-matic assay, according to the manufacturer’s protocol.
Immunohistochemical staining
Left ventricular tissues from the posterior muscle were rapidly removed, washed for 1 minute with 0.1 mol/L PBS (pH 7.4) and immersed in 4% paraformaldehyde in PBS containing 1% CaCl2 overnight. Hearts were serially sec-tioned in a sliding microtome coupled to a freezing unit for frozen slices of 10 μm thickness. According to the manufacturer’s protocol, anti ChAT staining was exam-ined by immunohistochemical analysis with the rabbit anti-rat ChAT antibody (1: 100 for 3 hours at 37 °C, PBS as negative control). Similar sections were also treated with a primary antibody specific for rabbit anti-rat SYN (1: 200), and rabbit anti-rat GAP-43 (1: 200) antibody. Fol-lowing this, sections were colored with strepta-vidin-biotin-peroxidase complex (SABC)- conjugated goat anti-rabbit IgG for 30 minutes, mounted and photo-graphed at 100 × magnification. In addition, sections were counterstained with hematoxylin-eosin. Five sec-tions from each rat were adopted for each index. Brown yellow grains in the cells indicated positive staining. The densities of GAP-43-positive and SYN-positive (numbers of nerve fibers/mm2) were determined.
Western blot detection of ChAT, SYN and GAP-43
The remaining left ventricular tissues were used to detect ChAT, SYN and GAP-43 by Western blot analysis. Briefly, cardiac tissue extracts were adjusted to the same protein concentration and 50 μg protein per lane was subjected to sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE). Following this, proteins were blotted onto 0.45-μm nitrocellulose membranes, blocked with 5% non-fat milk and incubated with a primary poly-clonal rabbit antibody (ChAT, SYN, GAP-43 and β-actin; 1: 500 dilution) overnight at 4 °C. The nitrocellulose membrane strips were rinsed in tris-buffered saline for 30 minutes, followed by incubation with horseradish per-oxidase (HRP)-conjugated goat anti-rabbit IgG secon-dary antibody (1: 2 000) for 1 hour at room temperature. The chemiluminescent signals (i.e. integral absorbance) were detected by SCION Image software (Scion Corpo-ration, USA). Relevant band intensities were quantified after normalization to the amount of β-actin protein.
Echocardiographic examination
Echocardiographic examination was performed by an investigator blinded to treatment allocation prior to, 2 and 4 weeks after adriamycin application. Following anes-thesia by intraperitoneal injection of 10% chloral hydrate, Wistar rats were placed in a prone position. Two-dimensional and M-mode echocardiograms were obtained at the level of the papillary muscles with an echocardiographic system. According to the American Society for Echocardiology leading-edge method[16], left ventricular dimensions were measured at least three consecutive cardiac cycles. Left ventricular ejection frac-tion (LVEF) was acquired and the mean value of LVEF < 45% was referred to as the standard of heart failure.
Main outcome measures
Myocardial NE content, ChAT, SYN and GAP-43 ex-pression.
Design, enforcement and evaluation
This study was designed by Hongliang Kong, Zhanquan Li and Guoxian Qi, and performed by Hongliang Kong and Yingjun Zhao. Hongliang Kong and Shumei Zhao provided new analytical tools and reagents. Hongliang Kong, Weiwei Zhang, Guiping Xu and Wenjun Hao analyzed experimental data. Hongliang Kong, Shumei Zhao, and Li Zhu drafted this paper. A blind method was employed throughout the whole process.
Statistical analysis
Experimental data were statistically processed using SPSS 12.0 software (SPSS, Chicago, IL, USA) and were expressed as Mean ± SD. One-way analysis of variance and Chi-square test, as well as linear regression analysis were adopted.

RESULTS

Effects of treatment on sympathetic excitation  
Myocardial NE content was significantly lower in the Akt-MSC group than in the s-MSC and control groups. Furthermore, they were significantly lower in the s-MSCs group than in the control group (P < 0.01). Myocardial NE contents were negatively correlated to LVEF (r = –0.64,  P = 0.015; Table 1, Figure 1)

Effects of Akt-MSCs on myocardial ChAT expression
Immunohistochemistry results demonstrated that ChAT-positive expression was observed in the Akt-MSCs, s-MSCs and control groups (Figure 2). There was no significant difference in ChAT expression between the Akt-MSCs and s-MSCs groups (P > 0.05), as determined by Western blot analysis. In contrast, the expression was significantly higher in the two treatment groups than the control group (P < 0.05; Table 1, Figure 3).
Effects of treatment on myocardial SYN expression
Immunohistochemistry results demonstrated that nerve fibers positive for SYN were observed in the posterior left ventricular wall myocardium in each group (Figure 2). In addition, the numbers of nerve fibers per mm2 that stained positive for SYN were significantly greater in the Akt-MSCs group than the s-MSCs and control groups  (P < 0.05). Furthermore, the numbers were significantly greater in the s-MSCs group than in the control group  (P < 0.05; Table 1). Semi-quantitative Western blot re-sults of myocardial SYN expression between two treat-ment groups were similar to immunohistochemistry re-sults (Table 1, Figure 3).
 

Effects of treatment on myocardial GAP-43 expres-sion
Immunohistochemistry results reveal that GAP-43-positive expression was shown in the posterior left ventricular wall myocardium in each group (Figure 2). In addition, the number of nerve fibers per mm2 that stained positive for GAP-43 was significantly greater in the Akt-MSCs group than the s-MSCs and control groups (P < 0.05). Furthermore, the numbers were significantly greater in the s-MSCs group than the control group (P < 0.05; Table 1). Semi-quantitative Western blot results of myocardial GAP-43 expression between two treatment groups were similar to immunohistochemistry results (Table 1, Figure 3).
Quantitation of experimental rats and LVEF value
At 2 weeks after adriamycin administration, six rats died of heart failure and other complications (mortality rate 15%). CHF rats exhibited asthenic and lethargic mani-festations, with LVEF value < 0.45. At 4 weeks, four rats died (one in Akt-MSCs group, one in s-MSCs group, and two in control group); however, there was no significant difference in the mortality rate among the three groups (P > 0.05). Compared with pre-treatment values, the LVEF was significantly higher after treatment in the Akt-MSCs and s-MSCs groups (P < 0.01). After treatment, the LVEF value was highest in the Akt-MSCs

group, followed by the s-MSCs and control groups, re-spectively (Figure 4).

DISCUSSION

Adriamycin, due to its irreversible cardiac side effects, which include cardiomyopathy and congestive heart fail-ure[14, 17-18], is the standard drug for establishing CHF models. The underlying mechanisms possibly include cardiac cell apoptosis[19-20], refractory inotropic support, dilated tubular membrane systems, myofibril dropout, upregulated inflammatory cytokines[17-18], and oxidative stress[21-22] mediated by MAPK signaling pathways[23]. In this study, we used CHF models to demonstrate that transplantation of Akt-MSCs and s-MSCs significantly improved heart function by elevating LVEF. Akt-MSCs were more effective and results indicated that trans-planted Akt-MSCs (or MSCs) have beneficial effects on myocardial function. There are a number of mechanisms by which they may produce these therapeutic effects. First, Akt-MSCs and MSCs may induce myocardial re-generation[24] and therapeutic angiogenesis[25-27], by dif-ferentiating into cardiomyocytes, vascular endothelial cells, and smooth muscle cells[27]. Second, they may also inhibit ventricular remodeling[27], by decreasing collagen deposition in the myocardium[27-28]. Third, they may pro-vide angiogenic, antiapoptotic and mitogenic factors[27, 29], by secreting large amounts of vascular endothelial growth factors, hepatocyte growth factor, adrenomedullin and insulin-like growth factor-1[25-33], which play an im-portant role in improving cardiac function. All of these mechanisms could be reinforced by the Akt gene[11-13]. However, the present study aims at elucidating the car-diac neuroprotective effects of MSCs transduced with Akt gene and therefore does not investigate all of the aforementioned effects. One of the main reasons for there being no significant difference in the mortality rate among the three groups is that the sample was not large enough to demonstrate whether Akt-MSCs and MSCs influence the survival rate of CHF rats. There has been a strong consensus that the over-activity of sympathetic tone is a characteristic of CHF which aggravates the adverse effects, progression and outcome of CHF [34], based on human and animal neurochemical studies. However, as one of the earliest features of neurohor-monal modulation in CHF, the pattern of activation of cardiac sympathetic nerves is fairly heterogeneous in CHF, based on several paradoxical findings. Some studies showed decreased density of the sympathetic innervation[35-36] and a declined rate of NE uptake during passage through the failing heart[37-38]. Results from the present study demonstrate that Akt-MSCs and s-MSCs significantly downregulate tissue NE level, which is strongly negatively correlated with LVEF. This is in ac-cordance with previous detailed kinetic analyses, show-ing that the release of NE into interstitial fluid is inversely correlated with left ventricular performance[39]. These results suggest that local Akt-MSCs and s-MSCs may relieve cardiac sympathetic activation. The following factors may contribute to downregulation of NE levels in the treatment groups. First, the plasma levels of NE de-creased with improved cardiac function, which is mainly attributable to improved cardiac remodeling[40]. Second, the local autocrine/paracrine response, such as the re-lease of nerve growth factor[25], regulates cardiac sym-pathetic nerve function by upregulating innervation den-sity, which plays a pivotal role in the transduction of sympathetic activation into the modulation of contractility and heart rate. Third, cardiac sympathetic nerve endings re-adjust the balance of NE storage/release/uptake ap-paratus, which is predominantly responsible for NE re-lease[41-42]. Akt-MSCs and s-MSCs may correct the de-cline of the cardiac sympathetic transmitter in the failing heart. In view of the reduction of cardiac sympathetic neurotransmitters relating to the increased interstitial NE in CHF[40], it is presumed that Akt-MSCs and s-MSCs may promote sympathetic nerve fiber sprouting. However, the mechanisms underlying nervous regeneration, me-diated by Akt-MSCs and s-MSCs need to be further examined. CHF is characterized by the withdrawal of parasympathetic tone[43-44] and augmented parasympa-thetic neurotransmission may play a key role in delaying the proceeding of CHF[45-47]. Therefore, it is necessary to identify the effects of Akt-MSCs and s-MSCs on the car-diac parasympathetic tone. As ChAT, an acetylcholine synthesizing enzyme, has been accepted as the most reliable marker protein for labeling cardiac cholinergic nerves[48]. In the present study, cholinergic nerve fibers were analyzed by means of ChAT staining and semi-quantitative analysis. Our findings demonstrate that the density of nerve fibers and the semiquantitative value were significantly higher in the Akt-MSC and s-MSC groups than the control group. This supports the hy-pothesis that MSCs improve heart function and may be related to augmented parasympathetic neurotransmis-sion; however, further studies are needed regarding how MSCs improve parasympathetic tone. The present re-sults demonstrate that both Akt-MSCs and s-MSCs could improve cardiac innervation of both sympathetic and parasympathetic fibers, by maintaining the balance of the intrinsic cardiac nervous system. This suggests that Akt-MSCs and s-MSCs may relieve and abolish the ab-normalities characterized by sympathetic over-activity and parasympathetic withdrawal[43-44] in the failing heart; however, it is difficult to ascertain how this detailed regulation is controlled. Nonetheless, in this study, we further elucidated the reasons for MSCs enhancing the density of nerve fibers.  
Although it is now almost universally agreed that SYN has no essential role in synaptic vesicle exocytosis[49], it remains one of the synapse-specific markers[50] as it is one of the most abundant proteins in synaptic vesicles, and is copiously expressed in the periphery of neurons. The present results also demonstrate that both Akt-MSCs and s-MSCs significantly strengthen the den-sity of SYN-positive fibers, suggesting anatomical re-connection between sprouting fibers and distal neurons, which further hints the development of the intrinsic car-diac neurons system.
GAP-43, localized in the presynaptic terminals and axons of cortical neurons[51-52], is an important regulator of axonal outgrowth and an intrinsic determinant of synaptic remodeling in increasing neuronal sprouting, which is expressed during the development of the nervous system[53]. The increased expression of GAP-43 may be beneficial to the promotion of axonal regeneration[53-54] and neuronal plasticity[55-56]. The present data revealed an obvious role in over-expressing Akt in MSCs, which suggests a more general role of Akt-MSCs in the control of neuronal outgrowth program. However, a means of improving the overexpression of GAP-43 in the failing heart remains undetermined.
Further studies are needed to address some limitations of the present study. First, the balance mechanism of cardiac autonomic nervous system, mediated by Akt-MSCs (including MSCs), is needed to confirm these results in vivo. Second, we did not check the differen-tiation of transplanted MSCs into neurons and whether Akt-MSCs are more effective than s-MSCs. Third, there is a possibility that the effects of the transplanted Akt-MSCs or s-MSCs may play a significant role in im-proving the autonomic nervous system by the promo-tion of endothelial differentiation or angiogenesis and other respects after MSC transplantation. Fourth, the safety of introducing Akt should been cautioned due to the tumorigenic potential of this pathway. Lastly, the presence of transplanted MSCs in the myocardium and other tissues should be determined.
In conclusion, the present results further support the role of MSCs in cellular therapy after CHF. Akt-1 gene-modified MSCs were more effective than simple MSCs in adjusting both parasympathetic activation and sympathetic activation, due to a fact that transplantation of Akt-MSCs further promotes the repair of injured heart function, compared with MSC transplantation alone. In addition, all effects may be mediated by nervous regeneration and nervous balance, in which GAP-43 possibly plays a considerable role.

REFERENCES

[1] Braunwald E, Bristow MR. Congestive heart failure: fifty years of progress. Circulation. 2000;102(20 Suppl 4):IV14-23.
[2] Aydin M, Altin R, Ozeren A, et al. Cardiac autonomic activity in obstructive sleep apnea: time-dependent and spectral analysis of heart rate variability using 24-hour Hoher electrocardiograms. Tex Heart Inst J. 2004;31(2):132-136.
[3] Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res. 2004;95:9-20.
[4] Amado LC, Saliaris AP, Schuleri KH, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A. 2005; 102(32):11474-11479.
[5] Tomita S, Mickle DA, Weisel RD, et al. Improved heart function with myogenesis and angiogenesis after autologous porcine bone marrow stromal cell transplantation. J Thorac Cardiovasc Surg. 2002;123:1132-1140.
[6] Gojo S, Gojo N, Takeda Y, et al. In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells. Exp Cell Res. 2003;288:51-59.
[7] Dai W, Hale SL, Martin BJ, et al. Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects. Circulation. 2005;112:214-223.
[8] Kong HL, Liu NN, Huo X, et al. Cell multiplication, apoptosis and p-Akt protein expression of bone mesenchymal stem cells of rat under hypoxia environment. J Nanjing Med Univ. 2007;21(4): 233-239.
[9] Kong HL, Zhang LQ, Cao SF, et al. Apoptosis and proliferation of rat bone marrow mesenchymal stem cells transfected by Akt gene under hypoxia. Zhongguo Zuzhi Huaxue yu Xibao Huaxue Zazhi. 2008;17(3):225-231.
[10] Noiseux N, Gnecchi M, Lopez-Ilasaca M, et al. Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation. Mol Ther. 2006;14(6):840-850.
[11] Gnecchi M, He H, Noiseux N, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J. 2006;20(6):661-669.
[12] Mirotsou M, Zhang Z, Deb A, et al. Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair. Proc Natl Acad Sci U S A. 2007;104(5):1643-1648.
[13] Teraoka K, Hirano M, Yamaguchi K, et al. Progressive cardiac dysfunction in adriamycin-induced cardiomyopathy rats. Eur J Heart Fail. 2000;2(4):373-378.
[14] Li SG, Mao XB, Zhang F, et al. A modified rat model of dilated cardiomyopathy induced by adriamycin. Zhongguo Bijiao Yixue Zazhi. 2006;16(7):415-418.
[15] Liang C, Rounds NK, Dong E, et al. Alterations by norepinephrine of cardiac sympathetic nerve terminal function and myocardial beta-adrenergic receptor sensitivity in the ferret: normalization by antioxidant vitamins. Circulation. 2000;102(1):96-103.
[16] Yang XP, Liu YH, Rhaleb NE, et al. Echocardiographic assessment of cardiac function in conscious and anesthetized mice. Am J Physiol. 1999;277: H1967-1974.
[17] Lefrak EA, Pitha J, Rosenheim S, et al. A clinicopathologic analysis of adriamycin cardiotoxicity. Cancer. 1973;32:302-314.
[18] Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med. 1998;339:900-905.
[19] Arola OJ, Saraste A, Pulkki K, et al. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res. 2000;60:1789-1792.
[20] Kumar D, Kirshenbaum LA, Li T, et al. Apoptosis in adriamycin cardiomyopathy and its modulation by probucol. Antioxid Redox Signal. 2001;3:135-145.
[21] Li T, Singal PK. Adriamycin-induced early changes in myocardial antioxidant enzymes and their modulation by probucol. Circulation. 2000;102:2105-2110.
[22] Siveski-Iliskovic N, Kaul N, Singal PK. Probucol promotes endogenous antioxidants and provides protection against adriamycin-induced cardiomyopathy in rats. Circulation. 1994;89:2829-2835.
[23] Lou H, Danelisen I, Singal PK. Involvement of mitogen-activated protein kinases in adriamycin-induced cardiomyopathy. Am J Physiol Heart Circ Physiol. 2005;288:H1925-1930.
[24] Shake JG, Gruber PJ, Baumgartner WA, et al. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg. 2002;73:1919-1925.
[25] Al-Khaldi A, Al-Sabti H, Galipeau J, et al. Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model. Ann Thorac Surg. 2003;75:204-209.
[26] Al-Khaldi A, Eliopoulos N, Martineau D, et al. Postnatal bone marrow stromal cells elicit a potent VEGF-dependent neoangiogenic response in vivo. Gene Ther. 2003;10:621-629.
[27] Nagaya N, Kangawa K, Itoh T, et al. Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation. 2005;112(8):1128-1135.
[28] Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation. 2001;104:1046-1052.
[29] Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet. 2002;360: 427-435.
[30] Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res. 2004;94:678-685.
[31] Nakamura T, Mizuno S, Matsumoto K, et al. Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF. J Clin Invest. 2000;106:1511-1519.
[32] Li Y, Takemura G, Kosai K, et al. Postinfarction treatment with an adenoviral vector expressing hepatocyte growth factor relieves chronic left ventricular remodeling and dysfunction in mice. Circulation. 2003;107:2499-2506.
[33] Tokunaga N, Nagaya N, Shirai M, et al. Adrenomedullin gene transfer induces therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia: benefits of a novel nonviral vector, gelatin. Circulation. 2004;109:526-531.
[34] Bristow M. Antiadrenergic therapy of chronic heart failure: surprises and new opportunities. Circulation. 2003;107: 1100-1102.
[35] Kaye DM, Vaddadi G, Gruskin SL, et al. Reduced myocardial nerve growth factor expression in human and experimental heart failure. Circ Res. 2000;86:E80-84.
[36] Himura Y, Felten SY, Kashiki M, et al. Cardiac noradrenergic nerve terminal abnormalities in dogs with experimental congestive heart failure. Circulation. 1993;88:1299-1309.
[37] Kaye DM, Lambert GW, Lefkovits J, et al. Neurochemical evidence of cardiac sympathetic activation and increased central nervous system norepinephrine turnover in severe congestive heart failure. J Am Coll Cardiol. 1994;23:570-578.
[38] Eisenhofer G, Friberg P, Rundqvist B, et al. Cardiac sympathetic nerve function in congestive heart failure. Circulation. 1996;93: 1667-1676.
[39] Grossman PM, Linares OA, Supiano MA, et al. Cardiac-specific norepinephrine mass transport and its relationship to left ventricular size and systolic performance. Am J Physiol. 2004;287: H878-888.
[40] Liang CS, Mao W, Iwai C, et al. Cardiac sympathetic neuroprotective effect of desipramine in tachycardia-induced cardiomyopathy. Am J Physiol Heart Circ Physiol. 2006;290(3): H995-1003.
[41] Kreusser MM, Buss SJ, Krebs J, et al. Differential expression of cardiac neurotrophic factors and sympathetic nerve ending abnormalities within the failing heart. J Mol Cell Cardiol. 2008; 44(2):380-387.
[42] Kristen AV, Kreusser MM, Lehmann L, et al. Preserved norepinephrine reuptake but reduced sympathetic nerve endings in hypertrophic volume-overloaded rat hearts. J Card Fail. 2006; 12(7):577-583.
[43] Rosenwinkel ET, Bloomfield DM, Arwady MA, et al. Exercise and autonomic function in health and cardiovascular disease. Cardiol Clin. 2001;19:369-387.
[44] Binkley PF, Nunziata E, Haas GJ, et al. Parasympathetic withdrawal is an integral component of autonomic imbalance in congestive heart failure: demonstration in human subjects and verification in a paced canine model of ventricular failure. J Am Coll Cardiol. 1991;18:464-472.
[45] Packer M. The neurohormonal hypothesis: a theory to explain the mechanism of disease progression in heart failure. J Am Coll Cardiol. 1992;20:248-254.
[46] Serra SM, Costa RV, Teixeira De Castro RR, et al. Cholinergic stimulation improves autonomic and hemodynamic profile during dynamic exercise in patients with heart failure. J Card Fail. 2009; 15(2):124-129.
[47] Androne AS, Hryniewicz K, Goldsmith R, et al. Acetylcholinesterase inhibition with pyridostigmine improves heart rate recovery after maximal exercise in patients with chronic heart failure. Heart. 2003;89(8):854-858.
[48] Mabe AM, Hoard JL, Duffourc MM, et al. Localization of cholinergic innervation and neurturin receptors in adult mouse heart and expression of the neurturin gene. Cell Tissue Res. 2006; 326:57-67.
[49] Evans GJ, Cousin MA. Tyrosine phosphorylation of synaptophysin in synaptic vesicle recycling. Biochem Soc Trans. 2005;33(Pt 6):1350-1353.
[50] Dlugos CA, Pentney RJ. Quantitative Immunocyto-chemistry of GABA and synaptophysin in the cerebellar cortex of old ethanol-fed rats. Alcohol Clin Exp Res. 2002;26:1728-1733.
[51] Benowitz LI, Shashoua VE, Yoon M. Specific changes in rapidly transported proteins during regeneration of goldfish optic nerve. J Neurosci. 1981;1:300-307.
[52] Benowitz LI, Perrone-Bizzozero NI, Finklestein SP, et al. Localization of the growth-associated phosphoprotein GAP-43 (B-50, F1) in the human cerebral cortex. J Neurosci. 1989;9: 990-995.
[53] Mahalik TJ, Carrier A, Owens GP, et al. The expression of GAP43 mRNA during the late embryonic and early postnatal development of the CNS of the rat: an in situ hybridization study. Brain Res Dev Brain Res. 1992;67(1):75-83.
[54] Namgung U, Routtenberg A. Transcriptional and post- transcriptional regulation of a brain growth protein: regional differentiation and regeneration induction of GAP-43. Eur J Neurosci. 2000;12(9):3124-3136.
[55] Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 1997;20: 84-91.
[56] Schmidt-Kastner R, Bedard A, Hakim A. Transient expression of GAP-43 within the hippocampus after global brain ischemia in rat. Cell Tissue Res. 1997;288:225-238.
 (Edited by Huang YJ, Hu LA/Song LP/Wang L)