Preserved left ventricular structure and function in mice ... - CiteSeerX

Am J Physiol Heart Circ Physiol 289: H1359 –H1365, 2005. First published May 13, 2005; doi:10.1152/ajpheart.01010.2004.

Preserved left ventricular structure and function in mice with cardiac sympathetic hyperinnervation Helen Kiriazis,1 Xiao-Jun Du,1 Xinheng Feng,1 Elodie Hotchkin,2 Tanneale Marshall,3 Samara Finch,3 Xiao-Ming Gao,1 Gavin Lambert,2 Julia K. Choate,4 and David M. Kaye3 1

Experimental Cardiology Laboratory, 2Human Neurotransmitter Laboratory, and Wynn Department of Metabolic Cardiology, Baker Heart Research Institute, Melbourne; and 4Department of Physiology, Monash University, Melbourne, Australia 3

Submitted 4 October 2004; accepted in final form 9 May 2005

Kiriazis, Helen, Xiao-Jun Du, Xinheng Feng, Elodie Hotchkin, Tanneale Marshall, Samara Finch, Xiao-Ming Gao, Gavin Lambert, Julia K. Choate, and David M. Kaye. Preserved left ventricular structure and function in mice with cardiac sympathetic hyperinnervation. Am J Physiol Heart Circ Physiol 289: H1359 –H1365, 2005. First published May 13, 2005; doi:10.1152/ajpheart.01010.2004.—Cardiacspecific overexpression of nerve growth factor (NGF), a neurotrophin, leads to sympathetic hyperinnervation of heart. As a consequence, adverse functional changes that occur after chronically enhanced sympathoadrenergic stimulation of heart might develop in this model. However, NGF also facilitates synaptic transmission and norepinephrine uptake, effects that would be expected to restrain such deleterious outcomes. To test this, we examined 5- to 6-mo-old transgenic (TG) mice that overexpress NGF in heart and their wild-type (WT) littermates using echocardiography, invasive catheterization, histology, and catecholamine assays. In TG mice, hypertrophy of the right ventricle was evident (⫹67%), but the left ventricle was only mildly affected (⫹17%). Left ventricular (LV) fractional shortening and fractional area change values as indicated by echocardiography were similar between the two groups. Catheterization experiments revealed that LV ⫾dP/dt values were comparable between TG and WT mice and responded similarly upon isoproterenol stimulation, which indicates lack of ␤-adrenergic receptor dysfunction. Although norepinephrine levels in TG LV tissue were approximately twofold those of WT tissue, TG plasma levels of the neuronal norepinephrine metabolite dihydroxyphenyglycol were fivefold those of WT plasma. A greater neuronal uptake activity was also observed in TG LV tissue. In conclusion, overexpression of NGF in heart leads to sympathetic hyperinnervation that is not associated with detrimental effects on LV performance and is likely due to concomitantly enhanced norepinephrine neuronal uptake. echocardiography; myocardial contraction; catecholamine; transgenic; nerve growth factor

hemodynamic abnormalities and mortality in patients (6, 26). Within this context, the therapeutic efficacy of ␤-blockers in patients with heart failure has been well documented (10, 17). The detrimental effects of enhanced adrenergic stimulation have been confirmed by recent studies on transgenic (TG) mice, which show that cardiac restricted overexpression of ␤1-, ␤2-, and ␣-adrenergic receptors (ARs) and Gs␣ protein all result in cardiomyopathy and premature death (9, 12, 21, 25, 28). Cardiac targeted overexpression of nerve growth factor (NGF), a neurotrophin that is essential for sympathetic neuronal differentiation, maturation, and survival (20, 27, 37), leads to sympathetic hyperinnervation of heart (18). It has been postulated (1, 18, 19) that this mouse model would also lead to heart failure or a cardiac dysfunction phenotype, a possibility that has not been investigated. However, although NGF plays a central role in promoting growth of sympathetic nerve dendrites, it is also important for synaptic function. Notably, once it is released, the majority of NE is cleared from the synaptic cleft via the NE transporter (11). NGF has been shown to increase uptake-1 activity in sympathetic nerves (39) and chromaffin cells (40) and to facilitate synaptic transmission in cultures of sympathetic neurons and cardiac myocytes (29). Hence, we hypothesize that although cardiac NGF overexpression leads to sympathetic hyperinnervation (18), it also promotes synaptic clearance of NE, and as such, adverse consequences after a prolonged adrenergic overstimulation, which is seen in failing heart, would not occur in this model. We have addressed this question by investigating heart morphology and in vivo function in mice with cardiac-specific overexpression of NGF. MATERIALS AND METHODS

CARDIAC SYMPATHETIC TONE IS enhanced under conditions of heart failure (13, 14). Prolonged and excessive sympathoadrenergic stimulation of heart leads to adverse biological consequences such as myocardial cell death, hypertrophy, and fibrosis (30). In the setting of heart failure, preferential activation of cardiac sympathetic nerves contributes to norepinephrine (NE) spillover from heart to plasma at 5–50 times the normal rate depending on the severity of the disease (14). Such enhanced NE spillover is due to both an increase in nerve firing rate and an attenuation in NE reuptake (14). Elevated plasma NE levels bear prognostic value (22) and have been linked to

Address for reprint requests and other correspondence: D. M. Kaye, Wynn Dept. of Metabolic Cardiology, Baker Heart Research Institute, P.O. Box 6492 St. Kilda Road Central, Melbourne, Victoria 8008, Australia (E-mail: [email protected]). http://www.ajpheart.org

Animals. These studies were approved by the local Animal Ethics Committee and conformed to guidelines set out in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Mice of either gender were studied at 5– 6 mo of age unless otherwise specified. TG mice (line 4, 8 –10 transgene copies) with NGF overexpression driven by a cardiac-specific ␣-myosin heavy-chain promoter (18) were studied in parallel to non-TG wild-type (WT) littermates. The colony was maintained in a DBA2J background. Echocardiography. In vivo heart function and chamber dimensions were assessed with two-dimensional targeted M-mode echocardiography while mice were under light anesthesia (6 mg/100 g ketamine, 1.5 mg/100 g xylazine, and 0.045 mg/100 g atropine ip) using a Hewlett-Packard Sonos 5500 ultrasonograph with a 15-MHz linear The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society

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array as described previously (38). Measurements of left ventricular (LV) end-diastolic dimension (EDD), end-systolic dimension (ESD), and wall thickness at end diastole were made from captured freeze frames of M-mode images by applying the leading-edge convention of the American Society of Echocardiography. Right ventricular (RV) and left atrial dimensions were measured from two-dimensional (2-D) images. Fractional shortening was calculated as [(EDD ⫺ ESD)/EDD] ⫻ 100%. To ensure that the LV functional comparison between WT and TG mice was not biased by a particular anesthetic, echocardiography was performed on a small cohort of animals using Avertin anesthesia (2.5 mg/10 g ip). Response to maximal ␤-agonist stimulation was also assessed in these mice 2 min after isoproterenol injection (3 ␮g/kg ip). Micromanometry. Blood pressure and LV contractile parameters were determined in closed-chest anesthetized mice (6 mg/100 g pentobarbitone and 0.06 mg/100 g atropine ip) using a 1.4-Fr Millar Mikro-Tip catheter as previously described (7). Values for LV ⫾dP/dt and heart rate (HR) were assessed at baseline and after intravenous infusion of isoproterenol (0.1– 6 ng/mouse). In addition, end-diastolic pressure and the time constant of LV relaxation (␶) were assessed at baseline. We calculated ␶ using the conventional exponential method and the logistic method (31). Curves were fitted to the pressure trace from the time of maximal ⫺dP/dt to a level 5 mmHg above the end-diastolic pressure of the next contraction; the correlation coefficient of each curve fit was ⱖ0.998. Isolated atrial experiments. To further investigate chronotropic properties, WT and TG mouse atria were isolated from ⬃3-mo-old mice and placed into an organ bath maintained at 37°C as previously described (5). The right and left atria were connected to an isometric force transducer using silk sutures. HR values were calculated from spontaneous beating rates. The preparation was left to equilibrate (for 45–90 min) until the HR did not change by ⬎10 beats/min for 20 min. Cumulative NE dose-response curves (10⫺8 to 10⫺4 mol/l) were subsequently obtained. Autopsy and histology. Atria, ventricles, and lungs were lightly blotted and weighed. Heart tissue and plasma were snap-frozen in liquid nitrogen and stored at ⫺80°C. Some whole hearts were fixed in 4% paraformaldehyde in phosphate-buffered solution (pH 7.4) for 12–18 h, subsequently processed, and fixed in paraffin blocks, and serial longitudinal 6-␮m sections were cut and stained with either hematoxylin and eosin or 0.1% picrosirius red (Polysciences). Catecholamine determination. Atrial and ventricular tissues were accurately weighed before being homogenized on ice in 0.4 ml of 0.4 mol/l perchloric acid that contained 0.01% EDTA. The homogenate was then rapidly centrifuged. Catecholamines were extracted from the perchloric acid supernatant and also from thawed plasma samples via alumina adsorption and were separated by high-performance liquid chromatography. Amounts were quantified by electrochemical detection according to previously described methods (24, 32). NE uptake. NE uptake studies were performed according to the methods of Percy et al. (35) with modifications. LV tissue strips (⬃20 mg) were obtained from 6-mo-old WT and TG mice and were preincubated for ⬃30 min at 37°C in Krebs-Henseleit solution (4 ml/well) that contained (in mM) 148 Na⫹, 4.0 K⫹, 1.8 Ca2⫹, 1.05 3⫺ Mg2⫹, 25 HCO⫺ 3 , 0.5 PO4 , 11 glucose, 0.027 EDTA, 0.4 ascorbic acid, 0.01 Ro 41-1049 (monoamine oxidase A inhibitor; Sigma RBI), 0.01 Ro 41-0960 (catechol-O-methyltransferase; Sigma RBI), and 0.03 corticosterone (an uptake-2 inhibitor; Sigma); some wells also contained 0.003 desipramine (an uptake-1 inhibitor; Sigma). This was followed by a 1-h uptake incubation with [3H]NE (0.25 ␮Ci/ml). We obtained [7-3H]NE (16 Ci/mmol) from NEN. After incubation, the LV strips were blotted, weighed, and placed in 3% trichloroacetic acid overnight before radioactivity was measured in scintillation fluid using a beta counter. In the presence of desipramine, [3H]NE uptake was reduced by ⬃70%. Levels of LV uptake-1 [in disintegrations/min (dpm)/mg] were estimated from the differences between [3H]NE contents in the absence and presence of desipramine. AJP-Heart Circ Physiol • VOL

Statistics. Results are presented as means ⫾ SE. Unless otherwise specified, comparisons between TG and WT mouse data were made using unpaired Student’s t-test, and dose-response curves were assessed using two-way repeated-measures ANOVA. Differences were considered significant at P ⬍ 0.05. When the ANOVA indicated a statistically significant difference, the Student-Newman-Keuls test was subsequently employed as a post hoc test. RESULTS

In vivo heart structure and function. Echocardiography revealed striking structural differences between TG and WT mouse hearts in vivo predominately in atria and right ventricles. Two-dimensional longitudinal (long-axis) images of hearts showed enlarged left atria and right ventricles in TG animals (Fig. 1A). The enlarged right ventricles were also clearly evident from the cross-sectional (short-axis) heart views (Fig. 1B). Specifically, RV EDD and the left atrium-toaorta diameter ratios were significantly increased in TG mice (Table 1). Conversely, LV dimensions were unchanged by overexpression of NGF (Fig. 1 and Table 1). In vivo RV and LV performance levels were not markedly different between TG and WT mice. Fractional shortening values as assessed from LV M-mode images were similar for the two genotypes (Table 1). RV fractional shortening values could be estimated from 2-D short-axis loops by measuring RV diameters at end diastole and end systole; these were also found to be similar between groups. Furthermore, fractional area changes, as determined by measuring the ventricular chamber areas at end diastole and end systole, were essentially identical for WT and TG mice for both left and right ventricles (Table 1). LV function values were also evaluated in a small cohort of animals anesthetized with Avertin (see below). From catheterization experiments, neither LV end-diastolic pressure (3.9 ⫾ 0.4 vs. 3.2 ⫾ 0.4 mmHg; n ⫽ 9 –12 mice) nor ␶ (exponential, 14.4 ⫾ 0.9 vs. 12.9 ⫾ 0.9 ms; logistic, 6.5 ⫾ 0.4 vs. 6.2 ⫾ 0.4 ms; n ⫽ 8 –10 mice) values were significantly altered between WT and TG mice, respectively. These data imply an absence of diastolic dysfunction in TG mice. Functional responses to ␤-agonists. Aortic systolic/diastolic pressures were similar between WT (n ⫽ 9) and TG (n ⫽ 8) mice at 91 ⫾ 5/59 ⫾ 4 vs. 93 ⫾ 5/61 ⫾ 5 mmHg, respectively. The postjunctional ␤-AR-mediated response was assessed in WT and TG mice (see Fig. 2A). Values for HR, ⫹dP/dt, and ⫺dP/dt were not significantly different between WT and TG mice under basal conditions. Furthermore, stimulation with isoproterenol revealed similar LV ⫾dP/dt dose-response curves for WT and TG animals (Fig. 2). There was a blunted HR response in TG mice that might have somewhat underestimated LV ⫾dP/dt values, which are rate-dependent parameters, at higher isoproterenol concentrations. Nevertheless, contractility values were similar between groups at the 1-ng isoproterenol dose where HR values were identical for WT and TG mice (Fig. 2A). LV function values under basal and maximal isoproterenolstimulated conditions (3 ␮g/kg ip) were also obtained upon echocardiographic examination of mice anesthetized with Avertin (n ⫽ 4 mice/group). First, LV fractional shortening values were not significantly different between WT and TG mice, which corroborates findings obtained when ketaminexylazine-atropine anesthesia was used. Second, as observed in

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Fig. 1. Altered chamber dimensions in transgenic (TG) hearts; evidence from echocardiography. Twodimensional longitudinal (A) and cross-sectional (B) echocardiographic images and M-mode images (C) from wild-type (WT) and nerve growth factor (NGF)overexpressing (TG) hearts are shown. In vivo images of TG hearts were clearly different from their WT counterparts and display enlarged left atria (LA) and right ventricles (RV); nonetheless, left ventricular (LV) chamber dimensions were relatively similar between the two groups. Ao, aorta; EDD, end-diastolic dimension; ESD, end-systolic dimension.

catheter experiments, the maximal chronotropic response to isoproterenol was blunted in TG mice (Fig. 2B). In vivo basal HR was measured while three anesthetic regimes were used including 1) ketamine, xylazine with atroTable 1. Echocardiography-derived cardiac dimensions and function Parameter

Wild-Type Mice*

Transgenic Mice†

Body mass, g Heart rate, beats/min LV end-diastolic dimension, mm LV mean diastolic wall thickness, mm 2-D RV end-diastolic dimension, mm Aortic diameter, mm Left atrial diameter, mm Left atrium-to-aorta ratio LV fractional shortening, % 2-D RV fractional shortening, % 2-D LV fractional area change, % 2-D RV fractional area change, %

25.3⫾1.5 355⫾12 4.04⫾0.08 0.72⫾0.01 2.96⫾0.11 1.42⫾0.03 1.93⫾0.06 1.37⫾0.06 37⫾2 29⫾2 48⫾3 35⫾2

25.1⫾0.9 371⫾6 3.97⫾0.09 0.76⫾0.02 3.87⫾0.28‡ 1.37⫾0.05 2.95⫾0.24‡ 2.12⫾0.15‡ 36⫾1 29⫾3 50⫾2 35⫾2

Values are means ⫾ SE; n ⫽ *10 or †10 or 11 mice. Transgenic mice had nerve growth factor overexpression in heart. 2-D indicates parameters were derived from two-dimensional echocardiography images. LV, left ventricular; RV, right ventricular; ‡P ⬍ 0.05 vs. wild-type. AJP-Heart Circ Physiol • VOL

pine (Table 1); 2) pentobarbitone with atropine (Fig. 2A); and 3) Avertin (Fig. 2B). Regardless which anesthetic was used, basal HR values were not significantly different between WT and TG mice. A difference was only observed in the presence of ␤-agonists, with the TG animals showing a blunted response. This was further investigated in vitro using atria isolated from WT and TG hearts (Fig. 2C). The increase in spontaneous beating rate in response to increasing NE concentrations was attenuated in TG atria. Autopsy and histological findings. Gross morphological differences between WT and TG mouse hearts were evident at autopsy and corroborated with findings from echocardiography. Specifically, TG hearts were characterized with markedly enlarged atria and right ventricles and had only mild hypertrophy of the left ventricles (degree of enlargement was as follows: right atrium ⬎ left atrium ⬎ right ventricle ⬎ left ventricle; Fig. 3, A, B, and D). Values for lung mass normalized for body mass were not significantly different between WT and TG mice (5.61 ⫾ 0.29 vs. 5.37 ⫾ 0.23 mg/g), which suggests the absence of pulmonary congestion in TG mice. Histological examination of TG hearts revealed the presence of substantial fibrosis in atria (as judged by positive picrosirius red staining) with less in right ventricles and only minor

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Fig. 2. Functional responses to ␤-adrenergic agonist. Isoproterenol dose-response curves demonstrate unchanged maximal rates of LV contraction (⫹dP/dt) and relaxation (⫺dP/ dt) in closed-chest cardiac catheterized NGF-overexpressing (TG) mice compared with WT mice (A). Heart rate (HR): isoproterenol dose-response curve was significantly blunted for TG vs. WT mice; #P ⬍ 0.05 for genotype times dose (two-way repeated-measures ANOVA). An attenuated HR response to maximal isoproterenol stimulation (hatched bars) was also evident in TG mice anesthetized with Avertin for echocardiographic studies (B). Values for basal fractional shortening (open bars) and HR were similar between the groups. *P ⬍ 0.05 compared with respective baseline (paired t-test); n ⫽ 4 mice/group. Additional evidence was obtained of blunted HR response in TG animals (C), this time in isolated atrial preparations exposed to increasing concentrations of norepinephrine ([NE]); *P ⬍ 0.05 compared with respective TG value; #P ⬍ 0.05 for genotype times dose (two-way repeated-measures ANOVA); n, no. of animals is shown.

changes in left ventricles (degree of fibrosis was as follows: right atrium ⬎ left atrium ⬎ right ventricle ⬎ left ventricle; Fig. 3, B and C). The upper part of the septum, which borders with the atria, was also affected. Fibrosis in the right ventricle appeared to be interstitial, whereas a different pattern was evident for the atria and affected septum in that the latter had highly nucleated areas (Fig. 3C). The striking effects of TG overexpression on atrial tissue prompted us to look at neonatal hearts. Hearts from 1-day-old TG mice were clearly discernible from their WT littermates,

particularly with regard to the appearance of atria (Fig. 3E), which suggests that the atrial changes largely occurred in utero. Heart and plasma catecholamine levels and neuronal uptake. The cardiac distribution of NE was inhomogeneous as follows: atria ⬎ right ventricle ⬎ left ventricle in WT hearts (Fig. 4A). A similar trend was seen in TG hearts except that NE was significantly increased compared with WT counterparts, and higher levels were observed in atria than ventricles (Fig. 4A). Tissue levels of dihydroxyphenylglycol (DHPG), which is an intraneural NE metabolite, and dihydroxyphenylacetic acid

Fig. 3. Gravimetric and histological findings. Hearts from 6-mo-old WT (A, left) and TG (A, right) mice are shown. Histological sections from hearts in A are shown as stained with picrosirius red (B). Areas stained red indicate the presence of collagen. This is particularly evident for TG right and left atria, right ventricle, and top of ventricular septum, whereas the rest of the left ventricle was largely unaffected. Note that there is a gradient of picrosirius red-positive staining from the base to the apex and also from the right to left chambers. Higher magnification of sections (C) stained with hematoxylin and eosin (top) and picrosirius red (bottom) from TG LV free wall (left), right ventricular free wall (middle), and right atrium (right). Bar graphs (D) show significant increases in total heart (H), left and right ventricle, and left and right atrial mass normalized for body mass for TG vs. WT mice. Hearts are shown (E) from 1-day-old (1 d) WT (left) and TG (right) mice. Bar graph shows heart mass (H) normalized for body mass (BM) in 1-dayold mice; *P ⬍ 0.05 (unpaired t-test); n, no. of animals in parentheses.

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Fig. 4. Catecholamine profile. NE contents of ventricular and atrial tissue from WT and TG mice are shown (A). WT mouse right and left atrial tissues were pooled (A). TG mice had significantly elevated NE levels compared with WT mice. Metabolites of NE and dopamine, dihydroxyphenyglycol (DHPG; B), and dihydroxyphenylacetic acid (DOPAC; C), were also increased in TG heart tissue. Plasma levels of epinephrine (E) and DHPG were elevated in TG mice compared with WT animals (D). *P ⬍ 0.05 compared with respective WT mouse value; #P ⬍ 0.05 compared with left atrium; n, no. of animals.

(DOPAC), a dopamine metabolite, were also assessed (Fig. 4, B and C). TG mouse hearts had increased levels of these metabolites, particularly in the atria. Plasma catecholamine levels were increased in TG mice (Fig. 4D). Moreover, plasma levels of DHPG were markedly increased (Fig. 4D). Because plasma levels of DHPG can serve as a marker of NE uptake (16), the data suggest that there was greater neuronal uptake and metabolism of NE in TG hearts. Myocardial uptake of [3H]NE was determined using LV muscle strips. LV uptake-1 (desipramine-sensitive)-mediated uptake of [3H]NE was greater in TG than in WT mice (3,532 ⫾ 109; n ⫽ 3 vs. 1,698 ⫾ 433 dpm/mg of tissue mass; n ⫽ 4 strips, respectively; P ⬍ 0.05). These findings are consistent with the catecholamine data and together imply enhanced NE neuronal uptake in LV myocardium of TG mice. DISCUSSION

At 6 mo of age, mice that overexpressed NGF in heart showed significant sympathetic hyperinnervation and remarkable changes in atria and right ventricles with substantial hypertrophy and fibrosis. These pathologies might have been expected considering the cardiomyopathic phenotype in TG mice due to overexpression of ␤1-AR (12), ␤2-AR (9, 28), ␣-AR (25), or Gs␣ protein (21) and long-term exposure to NE (3, 30). However, the NGF TG model differs from other cardiomyopathy models in that the structure and function of the left ventricle was basically normal and a blunted ␤-AR-mediated contractile response, which usually occurs under conditions of enhanced sympathoadrenergic stimulation or cardiac hypertrophy and failure (4, 15), was not present. The ␣-myosin heavy-chain promoter used to drive the NGF transgene is transcriptionally active in atria during embryonic development (18, 34); ventricular activation of this promoter only occurs after birth (34). The morphological abnormalities in the TG mice are most dramatic in the atria, and these AJP-Heart Circ Physiol • VOL

changes were evident in newborn animals, which indicates an “artificial phenotype” due directly to embryonic activation of the transgene. Earlier studies with this model have shown the presence of ectopic cells in atria and basal portions of heart that were likely derived from neural crest (1, 18). These cells do not exhibit contractile activity or membrane excitability and appear to be immature Schwann cells and ectomesenchymal-related cells (1). The large areas of positive picrosirius red staining (which is a marker of collagen) observed in TG mouse atria, at the base of ventricular septum, and in the right ventricle may be due to stimulatory effects of NGF on fibroblasts (33) in addition to the likelihood of NE-induced interstitial fibrosis (3). Our finding of preserved structure and function of the left ventricle in this model is of interest. Despite the evidence from catecholamine analysis of LV tissue that indicates sympathetic hyperinnervation, the morphological phenotype of the left ventricle is minor relative to the right ventricle and the atria, and LV function values at baseline and, more importantly, under ␤-adrenergic agonist stimulation were comparable to those of WT control mice. We consider this to be the likely “true” phenotype of NGF overexpression in vivo. Conversely, electrophysiological studies on isolated ventricular myocytes from 4- to 8-wk-old TG mice showed reduced peak inward calcium currents upon isoproterenol stimulation compared with WT mice (19). The same study showed unchanged total density of ␤1-AR, upregulation in ␤2-AR, and uncoupling of ␤-ARs from the adenylyl cyclase signaling pathway in TG mouse hearts (19). The present findings of intact functional responses to isoproterenol in TG mice in vivo by catheter and by echocardiography strongly indicate intact ␤-AR signaling in the left ventricles of TG mice. Because there is strong evidence that the response to sympathoadrenergic stimulation is largely mediated by ␤1-ARs (2, 8, 38), these results indicate that enhanced NGF activity leading to sympathetic hyperinnervation is not associated with ␤-AR dysfunction, a finding that

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differs significantly from that seen in diseased hearts. There was, however, a blunted chronotropic response to ␤-AR stimulation. The reason for this finding is not clear but seems intrinsic to the atria (as ex vivo experiments showed an analogous phenomenon) and may be related to histopathological abnormalities in atrial tissue compromising the function of pacemaker cells. Furthermore, it has been suggested (19) that TG mice may be predisposed to cardiac arrhythmias based on the finding of a significantly prolonged duration of action potential repolarization in isolated myocytes. However, functional data obtained from anesthetized TG animals did not reveal any arrhythmias. Catecholamine excess per se has detrimental effects on heart (3, 30). Furthermore, genetic models of enhanced adrenergic stimulation develop cardiomyopathic phenotypes (9, 12, 21, 25, 28). The present study is the first to investigate cardiac function in the NGF overexpression model. As stated above, there is no evidence of LV dysfunction even at 6 mo of age; also, the absence of lung congestion eliminates the possibility of any LV pump failure in these mice. Why does sympathetic hyperinnervation in this model not lead to the expected adverse effects on the left ventricle? Such disparity might be explained by the fact that NGF not only increases sympathetic nerve density but also enhances NE uptake (40). That this may be the case in the present study is supported by the findings in TG mice of increased plasma levels of DHPG and DOPAC, which are the intraneuronal metabolites of NE and dopamine, respectively. Furthermore, uptake-1 in LV tissue from TG mouse hearts was enhanced. Thus although the sympathetic drive to the heart of NGF TG mice is increased, the tonic activation of myocardial ARs is prevented by a concomitantly enhanced clearance of NE from the synaptic gap. These findings are in keeping with our hypothesis that the detrimental effects of an elevated sympathetic drive to the heart could be prevented if they are balanced by an increased uptake that functions as a braking mechanism to timely terminate AR activation. In conclusion, overexpression of NGF in heart leads to sympathetic hyperinnervation that is not associated with detrimental effects on LV structure and performance and is likely due to a concomitant enhancement of NE neuronal uptake. The observed phenotype of atrial and RV remodeling, hypertrophy, and fibrosis is unlikely due to enhanced cardiac sympathetic activity but is due directly to NGF overexpression during the developmental stage. Reduced expression of NGF has recently been observed in humans and with experimental congestive heart failure and is likely to be NE mediated (23, 36). Reduced NGF levels provide an explanation for the alterations in morphology as well as function of sympathetic neurons leading to uncontrolled NE spillover in congestive heart failure. We speculate that restoring NGF levels would be helpful in congestive heart failure by enhancing neuronal NE reuptake. Accordingly, conditional TG mouse models with cardiac-targeted overexpression of NGF offer an ideal approach for directly addressing this possibility. ACKNOWLEDGMENTS The authors sincerely thank Dr. Howard J. Federoff, University of Minnesota (source of NGF transgenic mice), and Dr. Jacqueline Hastings for excellent technical assistance. AJP-Heart Circ Physiol • VOL

GRANTS This work was supported by National Health and Medical Research Council of Australia Grant ID 225108 and a grant from the Atherosclerosis Research Trust (UK). REFERENCES 1. Andrade-Rozental AF, Rozental R, Hassankhani A, Spray DC, and Federoff HJ. Characterization of two populations of ectopic cells isolated from the hearts of NGF transgenic mice. Dev Biol 169: 533–546, 1995. 2. Bernstein D. Cardiovascular and metabolic alterations in mice lacking ␤1and ␤2-adrenergic receptors. Trends Cardiovasc Med 12: 287–294, 2002. 3. Briest W, Holzl A, Rassler B, Deten A, Leicht M, Baba HA, and Zimmer HG. Cardiac remodeling after long term norepinephrine treatment in rats. Cardiovasc Res 52: 265–273, 2001. 4. Bristow MR, Hershberger RE, Port JD, Gilbert EM, Sandoval A, Rasmussen R, Cates AE, and Feldman AM. ␤-Adrenergic pathways in nonfailing and failing human ventricular myocardium. Circulation 82: I12–I25, 1990. 5. Choate JK and Feldman R. Neuronal control of heart rate in isolated mouse atria. Am J Physiol Heart Circ Physiol 285: H1340 –H1346, 2003. 6. Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, and Rector T. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med 311: 819 – 823, 1984. 7. Du XJ, Autelitano DJ, Dilley RJ, Wang B, Dart AM, and Woodcock EA. ␤2-Adrenergic receptor overexpression exacerbates development of heart failure after aortic stenosis. Circulation 101: 71–77, 2000. 8. Du XJ, Fang L, Gao XM, Kiriazis H, Feng X, Hotchkin E, Finch AM, Chaulet H, and Graham RM. Genetic enhancement of ventricular contractility protects against pressure-overload-induced cardiac dysfunction. J Mol Cell Cardiol 37: 979 –987, 2004. 9. Du XJ, Gao XM, Wang B, Jennings GL, Woodcock EA, and Dart AM. Age-dependent cardiomyopathy and heart failure phenotype in mice overexpressing ␤2-adrenergic receptors in the heart. Cardiovasc Res 48: 448 – 454, 2000. 10. Eichhorn EJ and Bristow MR. Medical therapy can improve the biological properties of the chronically failing heart. A new era in the treatment of heart failure. Circulation 94: 2285–2296, 1996. 11. Eisenhofer G, Smolich JJ, and Esler MD. Disposition of endogenous adrenaline compared to noradrenaline released by cardiac sympathetic nerves in the anaesthetized dog. Naunyn Schmiedebergs Arch Pharmacol 345: 160 –171, 1992. 12. Engelhardt S, Hein L, Wiesmann F, and Lohse MJ. Progressive hypertrophy and heart failure in ␤1-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA 96: 7059 –7064, 1999. 13. Esler M, Jennings G, Lambert G, Meredith I, Horne M, and Eisenhofer G. Overflow of catecholamine neurotransmitters to the circulation: source, fate, and functions. Physiol Rev 70: 963–985, 1990. 14. Esler M, Kaye D, Lambert G, Esler D, and Jennings G. Adrenergic nervous system in heart failure. Am J Cardiol 80: 7L–14L, 1997. 15. Gilbert EM, Olsen SL, Renlund DG, and Bristow MR. ␤-Adrenergic receptor regulation and left ventricular function in idiopathic dilated cardiomyopathy. Am J Cardiol 71: 23C–29C, 1993. 16. Goldstein DS, Eisenhofer G, Stull R, Folio CJ, Keiser HR, and Kopin IJ. Plasma dihydroxyphenylglycol and the intraneuronal disposition of norepinephrine in humans. J Clin Invest 81: 213–220, 1988. 17. Goldstein S. Benefits of ␤-blocker therapy for heart failure: weighing the evidence. Arch Intern Med 162: 641– 648, 2002. 18. Hassankhani A, Steinhelper ME, Soonpaa MH, Katz EB, Taylor DA, Andrade-Rozental A, Factor SM, Steinberg JJ, Field LJ, and Federoff HJ. Overexpression of NGF within the heart of transgenic mice causes hyperinnervation, cardiac enlargement, and hyperplasia of ectopic cells. Dev Biol 169: 309 –321, 1995. 19. Heath BM, Xia J, Dong E, An RH, Brooks A, Liang C, Federoff HJ, and Kass RS. Overexpression of nerve growth factor in the heart alters ion channel activity and ␤-adrenergic signalling in an adult transgenic mouse. J Physiol 512: 779 –791, 1998. 20. Ip NY and Yancopoulos GD. The neurotrophins and CNTF: two families of collaborative neurotrophic factors. Annu Rev Neurosci 19: 491–515, 1996. 21. Iwase M, Bishop SP, Uechi M, Vatner DE, Shannon RP, Kudej RK, Wight DC, Wagner TE, Ishikawa Y, Homcy CJ, and Vatner SF.

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22. 23. 24. 25.

26.

27. 28.

29. 30. 31.

Adverse effects of chronic endogenous sympathetic drive induced by cardiac Gs␣ overexpression. Circ Res 78: 517–524, 1996. Kaye DM, Lefkovits J, Jennings GL, Bergin P, Broughton A, and Esler MD. Adverse consequences of high sympathetic nervous activity in the failing human heart. J Am Coll Cardiol 26: 1257–1263, 1995. Kaye DM, Vaddadi G, Gruskin SL, Du XJ, and Esler MD. Reduced myocardial nerve growth factor expression in human and experimental heart failure. Circ Res 86: E80 –E84, 2000. Lambert GW and Jonsdottir IH. Influence of voluntary exercise on hypothalamic norepinephrine. J Appl Physiol 85: 962–966, 1998. Lemire I, Ducharme A, Tardif JC, Poulin F, Jones LR, Allen BG, Hebert TE, and Rindt H. Cardiac-directed overexpression of wild-type ␣1B-adrenergic receptor induces dilated cardiomyopathy. Am J Physiol Heart Circ Physiol 281: H931–H938, 2001. Levine TB, Francis GS, Goldsmith SR, Simon AB, and Cohn JN. Activity of the sympathetic nervous system and renin-angiotensin system assessed by plasma hormone levels and their relation to hemodynamic abnormalities in congestive heart failure. Am J Cardiol 49: 1659 –1666, 1982. Lewin GR and Barde YA. Physiology of the neurotrophins. Annu Rev Neurosci 19: 289 –317, 1996. Liggett SB, Tepe NM, Lorenz JN, Canning AM, Jantz TD, Mitarai S, Yatani A, and Dorn GW 2nd. Early and delayed consequences of ␤2-adrenergic receptor overexpression in mouse hearts: critical role for expression level. Circulation 101: 1707–1714, 2000. Lockhart ST, Turrigiano GG, and Birren SJ. Nerve growth factor modulates synaptic transmission between sympathetic neurons and cardiac myocytes. J Neurosci 17: 9573–9582, 1997. Mann DL. Basic mechanisms of disease progression in the failing heart: the role of excessive adrenergic drive. Prog Cardiovasc Dis 41: 1– 8, 1998. Matsubara H, Takaki M, Yasuhara S, Araki J, and Suga H. Logistic time constant of isovolumic relaxation pressure-time curve in the canine left ventricle. Better alternative to exponential time constant. Circulation 92: 2318 –2326, 1995.

AJP-Heart Circ Physiol • VOL

H1365

32. Medvedev OS, Esler MD, Angus JA, Cox HS, and Eisenhofer G. Simultaneous determination of plasma noradrenaline and adrenaline kinetics. Responses to nitroprusside-induced hypotension and 2-deoxyglucose-induced glucopenia in the rabbit. Naunyn Schmiedebergs Arch Pharmacol 341: 192–199, 1990. 33. Micera A, Vigneti E, Pickholtz D, Reich R, Pappo O, Bonini S, Maquart FX, Aloe L, and Levi-Schaffer F. Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair. Proc Natl Acad Sci USA 98: 6162– 6167, 2001. 34. Palermo J, Gulick J, Colbert M, Fewell J, and Robbins J. Transgenic remodeling of the contractile apparatus in the mammalian heart. Circ Res 78: 504 –509, 1996. 35. Percy E, Kaye DM, Lambert GW, Gruskin S, Esler MD, and Du XJ. Catechol-O-methyltransferase activity in CHO cells expressing norepinephrine transporter. Br J Pharmacol 128: 774 –780, 1999. 36. Qin F, Vulapalli RS, Stevens SY, and Liang CS. Loss of cardiac sympathetic neurotransmitters in heart failure and NE infusion is associated with reduced NGF. Am J Physiol Heart Circ Physiol 282: H363– H371, 2002. 37. Sofroniew MV, Howe CL, and Mobley WC. Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci 24: 1217–1281, 2001. 38. Tan TP, Gao XM, Krawczyszyn M, Feng X, Kiriazis H, Dart AM, and Du XJ. Assessment of cardiac function by echocardiography in conscious and anesthetized mice: importance of the autonomic nervous system and disease state. J Cardiovasc Pharmacol 42: 182–190, 2003. 39. Wakade AR and Bhave SV. Facilitation of noradrenergic character of sympathetic neurons by co-culturing with heart cells. Brain Res 458: 115–122, 1988. 40. Wakade AR, Wakade TD, Poosch M, and Bannon MJ. Noradrenaline transport and transporter mRNA of rat chromaffin cells are controlled by dexamethasone and nerve growth factor. J Physiol 494: 67–75, 1996.

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