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Cardiovascular Research 1999 41(1):200-211; doi:10.1016/S0008-6363(98)00217-X
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

A soluble neuronal factor alters contractile function of ventricular myocytes without effect on troponin T isoform expression

Steven H. Greena, Rebecca M. Glovera, Penny A. Krummb, Timothy R. Wintersa and Dianne L. Atkinsb,*

aDepartment of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA
bDepartment of Pediatrics, University of Iowa, Iowa City, IA 52242, USA

* Corresponding author. Tel.: +1-319-356-3540; fax: +1-319-356-4693; e-mail: dianne-atkins@uiowa.edu

Received 6 October 1997; accepted 22 May 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The purpose of this investigation was to establish a model system to facilitate identification of the sympathetic neuronal factor(s) that promotes improved contractility in neonatal cardiac myocytes. Conditioned medium from PC12 cells with sympathetic phenotype served as the source of the neuronal factor. Methods: Contraction frequency, amplitude and velocity of cultured neonatal rat cardiac myocytes were measured by online video analysis. Interventions included in vitro sympathetic innervation, exposure to PC12 conditioned medium, neurotransmitters and antagonists. Metabolic activity was assayed by 2-deoxyglucose uptake. Troponin T isoform expression was analyzed by SDS–polyacrylamide gel electrophoresis. Results: Medium conditioned by neuronal PC12 cells induced contractility changes similar to those induced by in vitro sympathetic innervation. These effects of PC12 conditioned medium and innervation were not suppressed by adrenergic or muscarinic antagonists nor reproduced by neuropeptide Y or somatostatin. Neuronal PC12 conditioned medium but not chromaffin PC12 conditioned medium, increased metabolic activity of the myocytes as detected by [3H]-2-deoxyglucose, indicating that the effect was specific to the neuronal PC12 cells. The in vitro switch of troponin T isoform expression was not altered by exposure to PC12 conditioned medium. Conclusions: Increased contractile function induced by sympathetic innervation is reproduced by PC12 conditioned medium, but neither is mediated by sympathetic or muscarinic neurotransmitters. Troponin T isoform expression is not related to the contractility changes. This model system will allow identification of the factor(s).

KEYWORDS Cardiac myocytes; Contractile function; Neuronal factors; Sympathetic neurons; PC12 cells; Troponin T; Cell culture; Rats


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Sympathetic innervation in vitro has been shown to affect neonatal cardiac myocyte growth [1, 2], chronotropic response [3]and contractility [4]. These developmental responses can be demonstrated in a myocyte neuron co-culture system in which the myocytes are functionally innervated. This in vitro model is relevant to myocardial development in vivo where similar responses are modulated by sympathetic innervation. Friedman [5]has shown that the length tension relationship of intact hearts was increased in mature rabbits when compared to neonatal rabbits. Blockade of adrenergic receptors prevented this change, suggesting that the developmental regulation of contractility required neurotransmission. Since the onset of sympathetic neurotransmission is an early postnatal event, understanding the response of the heart to sympathetic innervation is important to understanding normal development and in understanding the response of the neonatal myocardium to adrenergic agonists if required to maintain the circulation. Understanding the relationship of sympathetic innervation to myocardial function also becomes important in later life when innervation is removed as in acute myocardial infarction or cardiac transplantation.

Lloyd and Marvin [4]have shown that the effects of innervation on myocyte contractility are mediated by a soluble factor produced by the neurons but different from the neurotransmitter. The present study supports a similar conclusion but more directly addresses the identity of the neuronal factor and its mechanism of action. Identification of the factor is complicated by the cellular heterogeneity of the primary cultures and by the small number of cells present in these cultures. Cell lines with sympathetic neuronal properties have been described and are potential sources of a neuronal factor. One such line is the widely used PC12 cell line derived from rat pheochromocytoma cells [6–8]. In the absence of nerve growth factor (NGF), these cells maintain a chromaffin cell-like phenotype but also display features of transformed cells and divide continuously in culture. In the presence of NGF, these cells stop dividing and acquire a cholinergic sympathetic neuronal phenotype, while losing chromaffin cell characteristics, including the capacity to synthesize catecholamines. We have previously shown that neuronally differentiated PC12 cells share with sympathetic neurons the ability to release a diffusible nonadrenergic factor that induces myocyte growth [9]. We now report that the increase in contractile function induced by sympathetic innervation is also induced by conditioned medium from the neuronally differentiated PC12 (nPC12) cells. nPC12 cells, which can be grown in large homogeneous populations, can serve as a source for purification of the factor.

Availability of an abundant source of the diffusible neuronal factor not only facilitates its eventual purification but also exposes all myocytes in a population to the factor. This allows investigation of biochemical correlates of neuron-induced physiological changes in cardiac myocytes. For example, several cardiac myocyte contractile proteins are known to undergo isoform switching during the course of normal development [10]. One of these, troponin T, has been shown to switch to the adult form from the embryonic form during the first week of life [11, 12], the same time that sympathetic neurotransmission is established in the neonatal rat. We show that this switch also occurs with the same timing in cultured cardiac myocytes. By treating cardiac myocyte populations with conditioned medium from nPC12 cell cultures we show that troponin T isoform switching occurs independently of exposure to diffusible neuronal factors and of induction of contractility changes.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Culture of primary cells
Primary cell cultures were prepared, as previously described [2, 9]from neonatal rat hearts obtained from the Wistar-Kyoto breeding colony at the University of Iowa Cardiovascular Center. The breeding animals were maintained according to the guidelines of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985) and the culture protocol was approved by the Animal Care and Use Committee of the University. Under aseptic conditions, the ventricular apices were surgically removed and placed in culture medium, composed of 85% MEM–Earle's salts, 15% horse serum, 4 mM L-glutamine, 20 µg/ml gentamicin, 20 mM HEPES buffer (pH 7.3), and 16 mM NaHCO3. After dissection was complete, the tissue fragments were rinsed for 5 min in potassium glutamate solution consisting of 140 mM K glutamate, 16 mM NaHCO3, 0.5 mM NaH2PO4, 25 mM HEPES (pH 7.3), 16.5 mM dextrose, and 0.014 mM phenol red. The tissue was minced into 2-mm fragments and then incubated with 1 mg/ml collagenase III for 20 min at 37°C. Enzymatic dispersion with trypsin (1 mg/ml) and mechanical dispersion was performed, followed by centrifugation as previously described [2, 9]. Total dissection and dispersion time was approximately 3 h. Following centrifugation, the pellet was resuspended in 10 ml of culture medium and incubated for 45 min to allow for fibroblast sedimentation. The ventricular cell suspension was plated at a density of 105 cells per ml onto 35-mm Nunclon tissue culture dishes or 2x104 per well in 24-well dishes, and then maintained in an incubator at 37°C, 5% CO2 and 95% humidity. Non-myocyte growth was inhibited by inclusion of 20 µM fluorodeoxyuridine and 10 µM uridine for 3 to 7 days.

Neuronal explants were prepared with separate instruments but simultaneously with the ventricular cells. Dissection of the neuronal tissue followed the removal of each ventricular apex. The paravertebral thoracolumbar sympathetic chains were removed from each pup, and placed in culture medium until dissection was complete. The neuronal tissue was finely minced (approximately 0.1 mm in diameter), placed by drops into the tissue culture dishes, and covered with growth medium, attaching to the culture dish within 4 h. The neuronal explants were incubated under the conditions described previously while myocyte preparation continued. Half of the ventricular cells were subsequently plated on dishes without explants. In both cases ventricular cell density was identical. Previous work [13]demonstrated that the myocytes do not exhibit functional innervation until 72 h in co-culture.

2.2 Verification of innervation
Innervation of individual myocytes was verified by light microscopic observation with modified differential interference optics prior to contractility measurements. This method correlates well with determination of innervation by a change in myocyte contraction frequency [3]or positive glyoxylic acid fluorescence of adrenergic neurons [13]. Adrenergic or muscarinic blockade was produced by the addition of propranolol 2 mM and prazosin 2 mM or atropine 2 mM in the culture medium after 48 h in culture. Adequacy of blockade was confirmed by the absence of contraction frequency change when the myocytes were acutely exposed to norepinephrine or acetylcholine [3, 4].

2.3 PC12 cell culture and preparation of conditioned medium
PC12 cell cultures were maintained as previously described [7]on collagenized dishes in 85% RPMI 1640, 5% fetal bovine serum and 10% donor horse serum. Such cells are chromaffin-like (cPC12 cells). Neuronal PC12 cells (nPC12) were prepared by replating cPC12 cells in RPMI 1640, 1% donor horse serum, and 50 ng/ml NGF then maintaining the cells in this medium for 14 days to ensure complete neuronal differentiation.

Conditioned medium was prepared from cultures of fully neuronally differentiated PC12 cells. Two days prior to the collection of the conditioned medium, the cells were fed with MEM, 1% donor horse serum, and 50 ng/ml NGF. The medium, conditioned for two days, was removed from the nPC12 cell dishes and filtered through a 0.4-µm filter to remove cells and debris. Glutamine and donor horse serum were then added to the nPC12 cell conditioned medium in order to make this medium equivalent to the control medium in all respects other than exposure to nPC12 cells. The conditioned medium was then mixed with an equal volume of fresh medium and added to non-innervated myocytes after they had been cultured for 48 h. The conditioned medium or control medium was removed every third day and replaced with fresh medium.

2.4 Contractility measurements
Contractile function of the ventricular cells was measured on the fourth day (96 h) after cell culture. Cells were studied in the dishes with the temperature maintained at 37°C by an air incubator. Spontaneously contracting myocytes were visualized by phase contrast microscopy using a 32x objective and a closed circuit video camera. The motion of the cell membrane or other high contrast organelle is measured with a video motion detector (Colorado Video, model 633, Boulder, Colorado) which monitors the position of the selected target along a selected raster line at the field rate of the video signal [4, 14]. The position of the targeted area of membrane is continuously monitored and converted to a voltage signal every 33 ms. The changes in this voltage signal which occur during cell contraction and relaxation are proportional to the contraction amplitude. The voltage signal from the video motion detector was filtered (7 Hz low pass) and calibrated to yield contraction amplitude in µm. The signal is electronically differentiated to provide velocity of contraction or relaxation (µm/s). Peak amplitude and peak velocity of contraction and relaxation are displayed on a physiologic monitor at a paper speed of 25 mm/s from which the rate of contraction can be measured. Recordings are made for five randomly selected spontaneously contracting cells in each culture dish. The target from each cell, which yields the greatest amplitude, was used for these analyses.

2.5 Assay of cellular metabolic activity by glucose uptake
Myocytes from newborn pups (<1 day old) were cultured for 2 days as described above. The culture medium was then exchanged for conditioned or control medium and the cells were maintained in these media for up to four days until the assay was performed. Cells maintained in these media for four days were refed with appropriate medium on the third day.

Cellular metabolic activity was assessed as 2-deoxyglucose [3H] uptake. Myocytes were washed with PBS/0.1% BSA and incubated in 1 ml PBS/0.1% BSA/0.5 µCi 2-deoxyglucose [3H] for 1 h. The cells were washed 4x with PBS/0.1% BSA, solubilized in scintillant, and internalized radioactivity was measured in a scintillation counter.

2.6 SDS–polyacrylamide gel electrophoresis and immunoblotting
Cells were washed twice in ice-cold PBS, and lysed in SDS–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer [15]. The lysates were heated to 100°C for 10 min and equal amounts of protein samples (5–10 µg) were applied to 12% SDS–PAGE minigels run according to Laemmle [15].

For visualization by immunoblotting, proteins were transferred to nitrocellulose at 4°C in 192 mM glycine/25 mM Tris base/20% (v/v) methanol. Protein transfers were blocked with 0.25% nonfat dry milk (NDM) in PBS overnight and then incubated with monoclonal antibody JLT12 (provided by Dr. Jim Lin, Department of Biological Sciences, University of Iowa) diluted 1:300 in PBS/NDM for 3 h at 37°C. The filters were then washed for 30 min with PBS/NDM and 0.05% Tween-20; then 3 washes for 20 min with PBS, 0.05% Tween-20; and finally 2 washes for 20 min with PBS. The filters were then incubated with alkaline phosphatase-conjugated goat anti-mouse antibody (Promega) diluted 1:500 in PBS/NDM. Washes were as for the primary antibody except that 0.1 mM MgCl2 was added to the final wash. The antibody was visualized by incubation in 0.33µg/ml nitroblue tetrazolium (Sigma/0.165 µg/ml BCIP Sigma/0.1 M Tris/0.1 M NaCl/5 mM MgCl2).

2.7 Statistical analysis
All measures of contractility were analyzed with one-way ANOVA and randomized block design to control for variation among cultures. The blocking variable was culture set. Post-hoc comparisons were made with Bonferroni's test for multiple comparisons [16]. Linear regression and correlation coefficients were used to examine the relationship between contraction frequency and amplitude. Statistical analyses were performed using SAS, Version 6.12, Cary, North Carolina. Data are reported as mean±standard error (SEM). Statistical significance was accepted at P<0.05.

2.8 Reagents
L-glutamine and donor horse serum were obtained from Hazelton Biologics. The trypsin was from Gibco Laboratories, the gentamicin from Sigma and the collagenase from Worthington. MEM–Earle's salts and KG solution were prepared by the Tissue Culture Hybridoma Facility, University of Iowa Cancer Center.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Regulation of myocyte contractility by factors secreted from neuronal cells
When myocytes were co-cultured with sympathetic neuronal explants, a subset of the myocytes became innervated. Innervated myocytes were easily identifiable by differential interference microscopy. Contractile function was measured after 96 h in co-culture, which is 48 h after functional innervation. In accord with previous studies [4], peak contraction amplitude (Fig. 1A), peak velocity of contraction (Fig. 1B) and peak velocity of relaxation (Fig. 1C) increased while contraction frequency (Fig. 1D) decreased. These observations confirm that sympathetic neurons directly promote increased myocyte contractile function, but do not distinguish between effects mediated by a diffusible factor as opposed to direct neuron–myocyte contact.


Figure 1
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  		Fig. 1 Contractility responses of cultured cardiac myocytes to innervation and neuronal factors. Peak contraction amplitude (A), peak velocity of contraction (B), peak velocity of relaxation (C), and contraction frequency (D) of cultured newborn rat ventricular myocytes treated under the following conditions: Ventricular myocytes under control conditions (CONT), innervated myocytes in neuron/myocyte co-cultures (INN), non-innervated myocytes in neuron/myocyte co-cultures (NON-INN), innervated myocytes in neuron/myocyte co-cultures treated with 2 mM prazosin and 2 mM propranolol (INN/B), non-innervated myocytes in neuron/myocyte co-cultures treated with 2 mM prazosin and 2 mM propranolol (NON-INN/B), ventricular myocytes treated with medium conditioned by neuronal PC12 cells (PC12), and ventricular myocytes treated with medium conditioned by neuronal PC12 cells and 2 mM prazosin and 2 mM propranolol (PC12/B), ventricular myocytes treated with acetylcholine (Ach), innervated myocytes treated with 2 mM atropine (Atr), and ventricular myocytes treated with PC12 conditioned medium and atropine. Measurements were from fifteen separate cultures: 61–102 cells for each condition. (* P<0.05 vs. CONT; + P=0.05 vs. CONT).

 
Many of the ventricular myocytes in the co-cultures never became innervated throughout the culture period. Contractile function of these cells was also assayed after 96 h in culture. The non-innervated cells also exhibited significant increases, relative to control myocytes cultured without neurons, in peak contraction amplitude (Fig. 1A), peak velocity of contraction (Fig. 1B), peak velocity of relaxation (Fig. 1C) and a significant decrease in contraction frequency (Fig. 1D). These changes were qualitatively similar to, but smaller than, those that occur in innervated cells. This indicates that neuron–myocyte contact is not necessary to improve contractile performance and suggests that the effect is mediated by a diffusible factor.

An obvious candidate for the diffusible factor is norepinephrine, the neurotransmitter released by these neurons. Although the concentration of norepinephrine is high at the neuron–myocyte synapse, the concentration in the culture medium is quite low [2]: norepinephrine is quickly oxidized in this culture medium, and no ascorbic acid was added to prevent oxidation. However, to conclusively demonstrate that the effects on the myocytes were not mediated by norepinephrine, neuron–myocyte co-cultures were exposed to 2 mM propranolol and 2 mM prazosin during the final 48 h of culture. As shown in Fig. 1, the effects of innervation or diffusible factors on myocyte contractility were not altered by complete adrenergic blockade: contractility changes apparent in innervated or non-innervated myocytes co-cultured with sympathetic neurons were not affected by propranolol or prazosin. We also tested the ability of atropine to block the effects of innervation. No effect was observed when atropine was added to the culture medium (Fig. 1).

An obstacle to future characterization of the diffusible factor is the relatively small number of sympathetic neurons that can serve as a source for its purification. We therefore assayed medium conditioned by nPC12 cells [8, 17]for similar activities. Like innervation, medium conditioned by nPC12 cells induced increases in peak contraction amplitude (Fig. 1A) and peak velocity of contraction (Fig. 1B), peak velocity of relaxation (Fig. 1C) and a decrease in contraction frequency (Fig. 1D) in cultured cardiac myocytes. Since PC12 cells become cholinergic upon neuronal differentiation [6], the conditioned medium should not contain norepinephrine. Consistent with this, we found that cardiac myocyte responses to nPC12 conditioned medium were not inhibited by adrenergic blockade (Fig. 1). However, neither did blockade with atropine alter the response to conditioned medium. The similarity of the conditioned medium effect to that of sympathetic innervation in all four measures of myocyte contractility provides strong evidence that cardiac myocyte contractility is regulated by a diffusible factor released by sympathetic neurons.

In addition to catecholamines, sympathetic neurons synthesize and secrete neuropeptides, including neuropeptide Y and somatostatin [18–20]. PC12 cells also are known to synthesize and release neuropeptide Y, with increased neuropeptide expression NGF treatment [21]. This raises the possibility that one of these neuropeptides might be responsible for the observed effects of neurons on cardiac myocyte contractility. However, treatment of cardiac myocytes with 100 nM neuropeptide Y or 10 nM somatostatin had no consistent effect on any aspect of contractility assayed (Fig. 2).


Figure 2
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  		Fig. 2 Contractility responses of cultured cardiac myocytes to neuropeptide V and somatostatin. Peak contraction amplitude (A), peak velocity of contraction (B), peak velocity of relaxation (C), and contraction frequency (D) of cultured newborn rat ventricular myocytes treated under the following conditions: Ventricular myocytes under control conditions (CONT), innervated myocytes in neuron/myocyte co-cultures (INN), non-innervated myocytes treated with 100 mM neuropeptide and for 48 (NPY 48) and 96 (NPY 96) h, non-innervated myocytes treated with 10 mM somatostatin for 48 (SOM 48) and 96 (SOM 96) h. Measurements were from 14 separate cultures: 60–80 cells for each condition. (* P<0.05 vs. CONT).

 
3.2 Relationship of contraction frequency and contraction amplitude
In adult cardiac preparations, positive and negative staircase phenomenon have been described which relate contraction frequency to force generation. Although staircase phenomenon are usually observed transiently, we tested the hypothesis that a negative staircase could explain the increased contraction amplitude which accompanied the decreased contraction frequency. Ventricular myocytes were treated with acetylcholine. Fig. 1 demonstrates that even though treatment with acetylcholine decreased contraction frequency, the other measures of contractile function remain unchanged. Linear regression analysis also demonstrated that contraction frequency was independent of contraction amplitude (Fig. 3). For control myocytes, the regression equation is y=0.012x+1.52, R2=0.005, P=0.48. The correlation coefficient was 0.007. The regression equation for innervated myocytes was y=0.015x+2.54, R2=0.005, P=0.61. The correlation coefficient was 0.003. The slopes of the regression lines for control and innervated myocytes are not different and there is poor correlation between contraction frequency and amplitude. The increases in contractile function occur independently of the decrease in contraction frequency.


Figure 3
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  		Fig. 3 Regression relationship between contraction frequency and amplitude of control and innervated myocytes. Contraction frequency and amplitude relationship for control ({triangleup}) and innervated ({blacksquare}) myocytes. For control myocytes, the regression equation is y=0.012x+1.52, R2=0.005, P=0.48. The correlation coefficient was 0.007. The regression equations for innervated myocytes was y=0.015x+2.54, R2=0.005, P=0.61. The correlation coefficient was 0.003.

 
3.3 Stimulation of cardiac myocyte glucose uptake by PC12 conditioned medium
Uptake of the non-metabolizable glucose analog 2-deoxyglucose (2-DG) has been widely used as a measure of cellular metabolism. We used this method to show that neuron-induced changes in cardiac myocyte contractility are accompanied by an increase in cellular metabolic activity. In most experiments, cardiac myocytes plated in control medium (without conditioned medium) showed a small increase, maximally 1.4- to 1.5-fold, in metabolic activity with time in culture (Fig. 4A, C). However, in some experiments, no significant increase in metabolic activity could be detected in cardiac myocytes plated in control medium (Fig. 4B).


Figure 4
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  		Fig. 4 Metabolic responses of cultured cardiac myocytes to medium conditioned by PC12 cells. (A) 2-deoxyglucose [3H] uptake (cpm per well) of newborn rat ventricular myocytes cultured in the absence of PC12 cell conditioned medium ({circ}), in the presence of medium conditioned by nPC12 cells ({blacksquare}), or in the presence of medium conditioned by cPCl2 cells ({blacktriangleup}). Four assays were performed on the first and fourth days after the addition of conditioned medium. * P <0.05 nPC12 vs. control day 4. (B) 2-Deoxyglucose [3H] uptake (cpm per well) of newborn rat ventricular myocytes cultured in the presence (filled symbols) of the absence (hollow symbols) of medium conditioned by neuronal PC12 cells showing the effects of adrenergic or muscarinic antagonists: no antagonists ({circ},bullet), 2 mM prazosin +2 mM propranolol ({square},{blacksquare}), 2 mM atropine,({triangleup},{blacktriangleup}). Four assays were performed on the first three days after addition of conditioned medium. * P<0.05 for all Day 1 innervated and nPCl2 vs. Day 2 innervated and nPC12. (C) 2-Deoxyglucose [3H] uptake (cpm per well) of newborn rat ventricular myocytes cultured in the absence of PC12 cell conditioned medium ({circ}), in the presence of medium conditioned by neuronal PC12 cells ({blacksquare}), in the presence of 100 nM neuropeptide Y ({triangleup}), or in the presence of 100 nM somatostatin ({triangledown}). Three assays were performed on the first and second days after addition of conditioned medium. In these experiments, a smaller well was used than in A and B with 30% of the number of myocytes plated. * P<0.05 for nPC12 on day 2 vs. other conditions on Day 2.

 
The addition of nPC12 conditioned medium to cardiac myocytes resulted in a 2- to 3-fold increase in 2-deoxyglucose uptake (Fig. 4). Conditioned medium from nonneuronally differentiated PC12 cells (chromaffin-like PC12 cells, cPC12 cells) was less efficacious than nPC12 conditioned medium. As shown in Fig. 4A, the myocytes exhibited a 1.4-fold increase in 2-deoxyglucose uptake in control medium, a 1.9-fold increase in cPC12 conditioned medium and a 3.1-fold increase in nPC12 conditioned medium. Thus, the ability to release the myocyte stimulating factor is largely a property of neuronal PC12 cells.

The density of cPC12 cells in the dishes from which conditioned medium was obtained was 5- to 10-fold greater than the density of the nPC12 cells (4500–9000 cells per mm2 vs. 900 cells per mm2). Thus, the effect of nPC12 cells on cardiac myocytes is considerably stronger than that of cPC12 cells on a per-cell basis. cPC12 cells secrete catecholamines into the medium [17]but PC12 cells become cholinergic as they assume the neuronal phenotype [6, 22]. Since conditioned medium from cPC12 cells has a relatively weak effect compared to the conditioned medium of nPC12 cells, metabolic stimulation of cardiac myocytes by PC12 cell conditioned medium is unlikely to be the result of catecholamines released by the PC12 cells. Furthermore, the effect of nPC12 conditioned medium is unaltered by adrenergic or muscarinic blockade (Fig. 4B) and is therefore due to neither adrenergic nor to cholinergic stimulation of the cardiac myocytes.

The possibility of regulation of myocyte metabolic activity by neuropeptide Y or somatostatin was also tested by this assay. The addition of 10 nM (not shown) or 100 nM concentrations of either neuropeptide Y or somatostatin had no significant effect on uptake of [3H]-2-deoxyglucose by the myocytes (Fig. 4C).

The nPC12 conditioned medium-induced changes in 2-deoxyglucose uptake occur over a time course similar to that of induced change in myocyte contractility. While in some instances a small change was detectable even 24 h after addition of conditioned medium, the change in level of 2-deoxyglucose uptake occurred largely between 24 and 48 h after addition of conditioned medium (Fig. 4). 2-Deoxyglucose uptake reached its maximal conditioned medium-induced level by 48 h after addition of conditioned medium and remained at this level for at least 96 h in conditioned medium (Fig. 4).

The increase in 2-deoxyglucose uptake is not due to an increase in the number of myocytes per well. Myocyte number declines by 6–10% in the first day following addition of nPC12 conditioned medium. After day 2, myocyte number declines at a rate of approximately 2% per day regardless of treatment (Fig. 5). Thus the observed increase in 2-deoxyglucose uptake is the result of increased metabolic activity of the myocytes.


Figure 5
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  		Fig. 5 Myocyte number in culture. Numbers of newborn rat ventricular myocytes cultured in the presence (bullet) or the absence ({circ}) of medium conditioned by neuronal PC12 cells. Numbers were determined by counting myocytes within five randomly chosen grids in each dish and multiplying this number by the ratio of the dish area to the area of the grids to obtain the number of cells in the entire dish. Three experiments were performed: in each experiment, 2–3 dishes were counted for each time point.

 
3.4 Troponin T isoform switching in vitro
Embryonic and adult cardiac myocytes differ in the troponin T isoform expression. The switch from the embryonic to the adult form during the first two postnatal weeks is due to a change in mRNA [23, 24]. In rat, the adult form is smaller than the embryonic form by ten amino acids and the two can be distinguished by size, with polyacrylamide gel electrophoresis [23–25]. Since this particular change is initiated over the same time period (postnatal days 1–4) as the innervation-induced contractility changes observed in cultured myocytes, we undertook to determine whether these two changes are related.

It was first necessary to document the time course of troponin T isoform switching in vitro. Thus, we assayed the expression of troponin T in cultured myocytes by immunoblotting with the JLT12 antitroponin T monoclonal antibody [26]. Fig. 6 shows that the switch occurred in vitro over a two week period, similar to that reported for myocytes in vivo [23]. The isoform switch is observable as an increase in the lower species of the troponin T doublet and a decrease in the upper species. Fig. 6 also shows that the rate of switching is largely the same in vitro as it is in vivo. Troponin T isoform ratios are approximately equal regardless of whether the myocytes were freshly obtained from pups of the 3, 6, 9, or 12 day old pups, or were obtained from newborn rats and then cultured for 3, 6, 9, or 12 days. The culture process does not alter the rate of isoform switching


Figure 6
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  		Fig. 6 Comparison of the time course of ventricular myocyte troponin T isoform switching in vivo and in vitro. Troponin T isoform expression in cultured myocytes determined by immunoblotting with the anti-troponin T monoclonal antibody JLT12. Cardiac myocytes taken from neonatal rats immediately after birth (postnatal day 0, P0) were cultured for 3–12 days (P3–P12). Cultured myocytes (C) were solubilized and used for determination of troponin T isoform expression at 3-day intervals, equivalent to postnatal ages P3, P6, P9 and P12 as indicated at the top of each lane. Each lane containing protein from cultured myocytes is paired with a lane containing protein from ventricular myocytes freshly isolated from rats of the same postnatal age (V).

 
3.5 Troponin T isoform switching by conditioned medium
Changes in in vitro myocyte contractile function, whether induced by innervation or by nPC12 conditioned medium, are concurrent with the initial switch from embryonic to adult troponin T isoform. In order to determine whether there is a causal relationship between expression of a particular troponin T isoform and contractile function, we assessed the effects of conditioned medium on troponin T isoform expression in cultured myocytes. Freshly isolated myocytes displayed primarily the embryonic form of troponin T. Expression of the adult isoform increased over the first four postnatal days in cultured myocytes: 4 day cultures displayed a pattern much like that of myocytes from P4 rat pups (Fig. 7). For comparison, the species corresponding to the lower molecular weight adult isoform is the single species that appears in myocytes isolated from P14 rat pups as has been described previously [23].


Figure 7
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  		Fig. 7 Troponin T isoform switching in myocytes cultured with neuronal conditioned medium. Troponin T isoform expression in cultured myocytes as determined by immunoblotting with the anti-troponin T monoclonal antibody JLT12. Freshly isolated myocytes were obtained from rats of postnatal ages 0 (P0), 4 (P4), and 14 days (P14) to show, respectively, expression of embryonic isoform alone, both isoforms, and adult isoform alone. Ventricular myocytes taken from P0 rats were cultured for 4 days (lanes 4–7) or 7 days (lanes 8 and 9) in the presence of the control medium (con), nPCl2 conditioned medium (npc), sympathetic neuron/myocyte-co-culture conditioned medium (sn/m), or medium conditioned by culture of dissociated neonatal rat superior cervical ganglion sympathetic neurons (sn).

 
Treatment of cardiac myocytes with nPC12 conditioned medium, which had a strong effect on contractility, had little or no effect on troponin T isoform expression (Fig. 7). Switch of troponin T isoform proceeded at the same rate in nPC12 conditioned medium-treated cultures as it did in the untreated controls (Fig. 7). We also examined the effects of two other types of conditioned medium. Lloyd and Marvin [4]have shown that conditioned medium from neuron/myocyte co-cultures affects myocyte contractility. Nevertheless, conditioned medium from neuron/myocyte co-cultures had no effect on the rate of troponin T isoform switching (Fig. 7). Similarly, conditioned medium from cultures of dissociated sympathetic neurons had no effect on the rate of troponin T isoform switching (Fig. 7). The switch from embryonic to adult troponin T isoform is nearly complete by the end of a week in culture, as it is in vivo: Fig. 7 shows that the extent of switching at this time is identical in control and nPC12 conditioned medium-treated myocytes.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
These studies support three major conclusions: (1) Sympathetic neurons can regulate cardiac myocyte contractility by release of a diffusible factor other than catecholamines. Addition of this factor to myocytes increases metabolic activity, as measured by 2-deoxyglucose uptake, concomitant with the improved contractile function. (2) The effect of sympathetic innervation on cardiac myocytes can be mimicked by medium conditioned by neuronal PC12 cells indicating that these cells are a potential source of the neuronal factor for the purpose of biochemical purification and characterization. (3) The switch in troponin T isoform expression, which is coincident in vivo with sympathetic innervation, occurs independently of diffusible neuronal factors. Moreover, the increase in contractile performance cannot be explained by a switching from embryonic to adult troponin T isoforms, despite the demonstration that the switch does occur in vitro.

4.1 Neuronal regulation of myocyte contractility
Two observations support our conclusion that the factor is diffusible. First, contractility is increased in myocytes co-cultured with sympathetic neurons but without neuron-myocyte contact and, second, similar changes in contractile function occur in myocytes treated with conditioned medium from the neuronal PC12 cell line.

It is possible that ‘noninnervated’ myocytes in the neuron–myocyte co-cultures are actually innervated by fine neurites that we could not detect by light microscopy. If so, this would account for the observation of neuron-induced contractility change without needing to postulate a diffusible factor. However, it is highly unlikely that cells identified as noninnervated by light microscopy are actually innervated. In two previous studies [1, 2]myocytes initially classified as innervated or noninnervated by light microscopy were subsequently examined in serial sections by electron microscopy for the purpose of determining their volumes. Of 93 cells classified as noninnervated by light microscopy, all proved to be noninnervated when examined by electron microscopy. This indicates that determination of innervation by light microscopy is accurate and that all or nearly all of the myocytes classified as noninnervated in this study are indeed noninnervated. Their response to neurons in co-culture therefore must require a diffusible neuronal factor.

Innervated myocytes display a much greater change in contractility than do their noninnervated counterparts in co-culture. This quantitative difference may be attributed to release of the factor at the neuromuscular junction in close contact with the innervated myocyte but distant from the noninnervated myocyte. Diffusion over the greater distances would presumably result in dilution of the factor. We attribute the quantitative differences between sympathetic conditioned medium and nPC12 conditioned medium to possible differences in concentration of the diffusible substance in the culture medium. PC12 cells are plated at a density of ~30 cells per mm2, while sympathetic neurons are plated at a density of ~3.5 cells per mm2. Thus, assuming that the synthesis and secretion rate are equal between the two neuronal cell types, the concentration of the factor would be approximately 9-fold higher in nPC12 conditioned medium than in sympathetic neuronal conditioned medium. It has been reported that direct contact between nerve and muscle is necessary to produce either innervation-induced increase in calcium channels [27], or changes in the alpha1-receptor [28]. However, other neural effects on skeletal and smooth muscle have been shown to occur in the absence of anatomic contact or even intact neurons [29–32]. Neurally derived soluble factors appear to regulate myocyte growth, isoform switching and cardiac myocyte contractile function.

To identify the diffusible factor, we evaluated several neurotransmitters and neuropeptides known to be produced by the sympathetic neurons or PC12 cells. The most obvious candidate was norepinephrine. Since adrenergic and muscarinic agonists have been shown to alter myocyte responsiveness and growth [3, 33], we tested the hypothesis that adrenergic or muscarinic stimulation promotes the changes in contractility induced in cardiac myocytes by sympathetic innervation in vitro. Previously, Lloyd and Marvin [4]have shown that adrenergic blockade does not affect contractility changes. We reproduced these results and extended the experiments to demonstrate that adrenergic blockade, with both alpha and beta adrenoceptor inhibitors failed to prevent the neurally induced changes in non-innervated cells in the co-culture dishes. Similarly, adrenergic blockade had no effect on contractility changes produced by nPC12 conditioned medium, although such conditioned medium would not have been expected to contain significant amounts of catecholamines [6, 22]. nPC12 cell conditioned medium may contain acetylcholine, however, atropine does not block the effects of nPC12 cell conditioned medium indicating that acetylcholine does not mediate the effect on the myocytes. Thus, the data presented here demonstrate that a neuronal factor other than the catecholaminergic or cholinergic neurotransmitter regulates myocyte contractility and that these neurotransmitters are not necessary for the effect. We have reported comparable results on myocyte growth regulation by a diffusible factor [9].

While neuropeptide Y alone may mimic some effects of sympathetic innervation on cultured rat ventricular myocytes [34], the contractility and metabolic changes described here are unaffected by neuropeptide Y. Somatostatin was equally ineffective at reproducing the effects of innervation or nPC12 conditioned medium. The identity of the released neuronal factor remains unknown. {alpha}-adrenergic-, β-adrenergic-, muscarinic-, neuropeptide Y-, and somatostatin-mediated stimulation of the cardiac myocytes can be ruled out by these studies. CGRP, another candidate neuropeptide, is not made by PC12 cells (A. Russo, personal communication) and therefore cannot be responsible for the effect. The medium in which neuronal PC12 cells are grown contains NGF but it is unlikely that NGF is the cause of physiological changes in conditioned medium-treated myocytes: NGF alone has no effect on 2-deoxyglucose uptake by myocytes (R.M. Glover and S.H. Green, unpublished observations). Furthermore, it is unlikely that significant amounts of NGF remain in the medium in which PC12 cells have been cultured for two days [35].

4.2 Troponin T isoform expression
In these studies, myocytes are cultured under conditions in which spontaneous contractions are maintained. Such conditions are likely to provide better model systems for studying changes in gene expression during myocyte development than conditions in which contractions do not occur. McDermott and Morgan [36]have shown that rates of mRNA and protein synthesis are reduced in quiescent myocytes. Samarel et. al. reported accelerated myosin heavy chain degradation with contractile arrest [37].

In cultured contracting rat myocytes, we observe changes in troponin T isoform expression with a time course similar to that observed in the rat in vivo [23]. Since, in some cases, we can detect a small amount of the adult isoform in P0 pups, we presume that initiation of the switch is a prenatal event. However, upon initiation of this program, transfer of the myocytes to culture does not alter its progression. Progress through the program of troponin T isoform switching is therefore independent of innervation or stimulation by nPC12 conditioned medium.

Most importantly, timing of the troponin T isoform switch is not affected by nPC12 cell conditioned medium. This dissociates the neuronal effect on cardiac myocyte contractile function from changes in troponin T isoform expression. Whatever physiological role troponin T isoform switching plays, it is unlikely to be directly related to the neuron-induced improved contractility. The role of troponin T isoform switching in cardiac development remains unclear.

In addition to the contractility changes addressed here, sympathetic innervation produces myocyte growth [1, 2, 9, 38]. These growth changes are also mediated by a soluble factor other than catecholamines, acetylcholine or one of the neuropeptides discussed above. Although {alpha}1-adrenergic stimulation induces myocyte growth under certain culture conditions, another neuronal factor does also [33]. Moreover, the growth-promoting activity is also found in medium conditioned by neuronal but not non-neuronal nPC12 cells [9]. While multiple factors may mediate the contractility, metabolic changes, and the growth response, it is possible that a single neuronal factor mediates all of these responses. An interesting possibility is that the neuronal factor causes the contractility changes directly and the growth is an indirect response to the increased contractile function. Because the myocytes are contracting while attached to the culture dish, the myocytes are presumably under mechanical tension. Stretching has been shown to induce myocyte growth in vitro, specifically via an autocrine mechanism involving angiotensin {Pi} [39]. Activation of such a mechanism by neuronal factors can be determined using the in vitro model described here.

We have initiated studies directed toward the identification of possible molecular correlates of contractility changes. Such studies are greatly facilitated by the observation that an easily-obtained conditioned medium can mimic the effects of innervation on myocytes. This makes identical treatment of myocytes possible in the entire population in contrast to innervation that is restricted to only a subset of the myocytes. Thus biochemical changes occurring concomitant with contractility changes are identifiable.

Time for primary review 22 days.


   Acknowledgements
 
Supported by grants R01-HL43173 (DLA and SHG) and R01-DC02961 (SHG) from the National Institutes of Health, 96-013680 (DLA) from the American Heart Association and the University of Iowa Diabetes and Endocrinology Research Core, NIH grant DK25295.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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