Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Regulation of voltage-gated K+ channel genes represents an important mechanism for modulating cardiac excitability. Here we demonstrate that expression of two K+ channel mRNAs is reciprocally controlled by cell-cell interactions between adult cardiac myocytes. It is shown that culturing acutely dissociated rat ventricular myocytes for 3 h results in a dramatic downregulation of Kv1.5 mRNA and a modest upregulation of Kv4.2 mRNA. These effects are specific, because similar changes are not detected with other channel mRNAs. Increasing myocyte density promotes maintenance of Kv1.5 gene expression, whereas Kv4.2 mRNA expression was found to be inversely proportional to cell density. Conditioned culture medium did not mimic the effects of high cell density. However, paraformaldehyde-fixed myocytes were comparable to live cells in their ability to influence K+ channel message levels. Thus the reciprocal effects of cell density on the expression of Kv1.5 and Kv4.2 genes are mediated by direct contact between adult cardiac myocytes. These findings reveal for the first time that cardiac myocyte gene expression is influenced by signaling induced by cell-cell contact.
voltage-gated K+ channel; rat ventricular myocyte; gene expression
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INTRODUCTION |
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LONG-TERM CHANGES IN cellular excitability can be generated by regulating the expression of K+ channel genes. For example, extensive studies in pituitary cells have demonstrated that regulation of Kv1.5 K+ channel gene transcription results in rapid changes in expression of voltage-gated K+ current (13, 27, 29). The first demonstration of regulation of cardiac K+ channel mRNA and protein expression was found in response to glucocorticoids (14, 28). Several studies have also identified modified cardiac K+ channel mRNA expression with animal models of hypertension and myocardial infarction (10, 17, 30) and with changes in thyroid hormone (23). Thus K+ channel gene expression in cardiac tissue is not static. Rather, it can be altered in response to numerous stimuli.
In contrast to the growing literature on changes in cardiac K+ channel gene mRNA in vivo, information concerning direct regulation of K+ channel gene expression in isolated adult cardiac myocytes is lacking. Regulation of neonatal myocyte K+ channel mRNAs has been demonstrated for members of the Kv1 subfamily in response to KCl, BAY K 8644, 12-O-tetradecanoylphorbol 13-acetate, or cAMP (17, 19). However, it is not clear whether similar control of channel genes occurs in adult cells. In fact, the expression pattern of cardiac K+ channel mRNAs changes during development. For example, rat Kv1.4 message levels are relatively high in the ventricle of neonates and very low in adult (32). In contrast, cardiac Kv4.2 increases during development (22, 32). Because our previous studies on cardiac channel expression were performed with adult animals, we set out to demonstrate direct regulation of K+ channel genes in isolated adult cardiac myocytes for the first time.
In this report, we show that expression of Kv1.5 and Kv4.2 K+ channel mRNAs in cultured adult rat ventricular myocytes is controlled by cell density. Despite the fact that these genes are reciprocally regulated, both effects are mediated by cell-cell contact between ventricular myocytes. This constitutes the first demonstration that signaling induced by cardiac myocyte-myocyte contact affects gene expression. This form of regulation could become significant following myocardial infarction and other pathological conditions that affect contact between myocytes.
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MATERIALS AND METHODS |
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Isolation of ventricular myocytes. Female Sprague-Dawley rats (195-225 g) were obtained from Zivic-Miller Laboratories, Hilltop Lab Animals, or Charles River Laboratories. All chemicals and drugs used in this study were obtained from Sigma Chemical unless noted otherwise. For all experiments shown, rats were injected intraperitoneally with 50 mg/kg dexamethasone in sesame oil to increase Kv1.5 gene expression (28). However, ventricular myocytes isolated from naive animals behaved in a quantitatively similar fashion to those isolated from dexamethasone-injected rats, indicating that this treatment had no impact on the effects described in this report (see RESULTS). All figures in this study show results from dexamethasone-injected rats.
The protocol used for the acute dissociation of cardiac myocytes from whole ventricles has been described previously (14). Briefly, animals were anesthetized with Metofane (Pitman-Moore), and hearts were excised and placed in ice-cold solution 1 (final concentrations, in mM: 5.4 KCl, 0.82 MgSO4, 120 NaCl, 1.0 NaH2PO4, 1.7 MgCl2, 2.0 L-glutamine, 4.4 NaHCO3, 21.2 HEPES, 1.5 KH2PO4, 8.0 NaOH, and 11.6 glucose, with 1× essential vitamins and 1× essential amino acids). After fat and lung tissue was trimmed off, the ascending aorta was cannulated and the heart was hung from a Langendorff perfusion apparatus. The perfusion was conducted in a 37°C warm room. The heart was perfused first with solution 2 (solution 1 supplemented with 0.1 µM insulin and an additional 5.0 mM MgCl2 and 1.0 mM NaOH) and then with solution 3 [solution 2 supplemented with 0.06% collagenase (type II, Worthington Biochemical) and an additional 0.01 mM CaCl2]. The two solutions used to perfuse the heart were continuously bubbled with 95% O2-5% CO2 for the entire perfusion period. Approximately 16 min after the beginning of perfusion with the collagenase-containing solution, the lower three-fourths of the heart, containing right and left ventricular tissue, was removed and returned to room temperature in a 60-mm culture dish containing 10 ml high-K+ solution (final concentrations, in mM: 40 KCl, 70 KOH, 20 KH2PO4, 20 taurine, 50 monopotassium glutamate, 10 HEPES, 0.5 EGTA, and 3.0 MgCl2, with 1 mg/ml BSA). The ventricular tissue was then cut into small pieces and triturated with a fire-polished Pasteur pipette, and the sample was poured over a 200-µm nylon mesh into a 15-ml centrifuge tube. Cells were washed twice with solution 4 (solution 1 supplemented with 5 mg/ml BSA, 1.25 mg/ml taurine, 5.0 mM MgCl2, 1.0 mM NaOH, and 0.01 mM CaCl2). This procedure yields ~9 × 106 ventricular myocytes. For experiments utilizing fixed myocytes, myocytes were isolated as above. As the sample (cells in 2.5 ml of solution 4) was vortexed, 7.5 ml 4% paraformaldehyde in PBS were added to the tube. The sample was vortexed for 2 min, and then cells were washed twice with 0.1% glycine in PBS and once with solution 4. Fixed myocytes were maintained at room temperature in this solution until live myocytes were generated (not longer than 4 h). Ventricular myocytes treated in this manner were indistinguishable from live myocytes. Cardiac myocytes were cultured at 37°C in a 5% CO2-humidified air incubator in medium 199 with 25 mM HEPES, in the absence of L-glutamine. Unless noted otherwise, the yield of cells from a single heart was divided equally into three cultures. See Figs. 1-8 for further details.mRNA measurements.
Total RNA was isolated according to the procedure of Chomczynski and
Sacchi (5). mRNA abundance was determined by Northern blot
hybridization (1, 27, 28, 29) and RNase protection assays (RPA) (1, 14,
27, 28, 30). For Northern blot hybridization, total RNA (15 µg) was
separated in 1% formaldehyde-agarose gels. RNA was then transferred to
GeneScreen (DuPont NEN) via capillary action and ultraviolet
cross-linked. Membranes were prehybridized at 42°C in 50%
deionized formamide, 0.25 M sodium phosphate (pH 7.2), 0.25 M NaCl, 1 mM EDTA, 7% SDS, 0.1% sodium pyrophosphate, and 0.15 mg/ml
heat-denatured salmon sperm DNA. Hybridization was
performed overnight in the same solution utilized for prehybridization.
cDNA probes were synthesized with the Boehringer Mannheim random primer
DNA labeling kit according to the protocol supplied by the
manufacturer, heat denatured, and then added to the prehybridization
buffer at a concentration of 3 × 106
counts · min1 · ml
1.
The Kv1.5 probe, corresponding to nucleotides 6-2136, was
generated from the Bgl
II/Hind III fragment of the Kv1 cDNA
(26). The Ca2+ channel probe
(rbC-1; Ref. 25) was synthesized from the carboxy-terminalmost fragment
generated by an EcoR I digest
(nucleotides 5322-7853). The cyclophilin probe was synthesized
from Pst I-linearized p1B15 (6). The
KvLQT1 cDNA was previously isolated by our laboratory (30) (GenBank no.
U92655). The whole insert was liberated by
Apa
I/Sac I digestion and used for probe
synthesis. Membranes were washed as described previously (27).
Autoradiographic exposures of X-ray film varied in length and
temperature. For densitometric analysis, exposed X-ray films were
scanned into DeskScan II (version 2.3, Hewlett-Packard) using a
Hewlett-Packard ScanJet IIC scanner. Densitometric analysis was
performed on channel mRNA signals that were within the linear range of
the film and had cyclophilin signals that also met this requirement.
After background subtraction, channel mRNA signals were normalized to
the background-subtracted cyclophilin signal. Normalized signals from
experimental groups were expressed as a percentage of the signal
obtained in the control. A Student's
t-test was performed to determine the
level of significance. Values were considered significantly different
when P
0.05.
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RESULTS |
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Rapid downregulation of Kv1.5 mRNA expression was observed when
myocytes isolated from whole ventricular tissues were cultured in
100-mm dishes (Fig.
1). Experiments with two
animals revealed a threefold decrease in Kv1.5 mRNA abundance in
ventricular myocytes cultured for 3 h (data not shown). Studying this
phenomenon was difficult, however, because of the low level of Kv1.5
message. Thus we injected animals with dexamethasone to increase Kv1.5 mRNA expression in the ventricles (28). When ventricular myocytes from
these animals were cultured for 3 h, an 86 ± 3% loss in Kv1.5 mRNA
was observed (n = 13). Consequently,
dexamethasone-injected rats were employed to generate all the figures
of this study. Fetal bovine serum (10%), laminin (10 µg/ml),
dexamethasone (1-20 µM), 8-bromoadenosine
3'5'-cyclic monophosphate (1 mM), and
3,5,3'-triiodothyronine (T3;
5 µM) each failed to prevent the loss of Kv1.5 message
(data not shown). The effect of culturing on Kv1.5 mRNA levels could not be attributed to poor cell viability. First, the effect was found
to be specific: mRNA levels for the KvLQT1 channel
(n = 6) and the
1C subunit of the L-type
Ca2+ channel
(n = 3) did not decrease following a
3-h incubation (Fig. 1B). KvLQT1 and
1C mRNA abundances at this time
were 102.9 ± 9.8 and 148.7 ± 39.1% of control, respectively.
Furthermore, inducible nitric oxide synthase mRNA increased when
ventricular myocytes were cultured for 24 h with interleukin-1
(n = 4), as expected (2). In addition,
Kv4.2 mRNA levels were slightly increased by
T3 (data not shown). Hence, these
results indicate that viable isolated adult rat ventricular myocytes
cannot maintain Kv1.5 gene expression under standard culture
conditions.
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Dissociation and culturing on dishes separate myocytes. However, cardiac myocytes spontaneously pellet in culture medium when they are stored in 15-ml conical tubes. Unexpectedly, ventricular myocytes cultured under the latter conditions maintained elevated Kv1.5 mRNA levels for 3-6 h (Fig. 2A, see also Fig. 4; n = 5). Because the formation of a pellet represents a condition of maximal cell density, the relationship between cell density and Kv1.5 mRNA levels was examined. Downregulation of Kv1.5 gene expression was found to decrease when a constant number of myocytes were plated in smaller vessels (Fig. 2B; n = 2). Furthermore, when cells were plated in 60-mm dishes, normalized Kv1.5 mRNA expression was proportional to cell number (Fig. 2C; n = 2). In contrast, reducing cell number does not have a large effect when cells are allowed to pellet (data not shown). These results indicate that maintenance of Kv1.5 gene expression is proportional to cell density.
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The effect of cell density could be mediated by diffusible paracrine factors or cell surface molecules that respond to cell-cell contact. To test for the presence of a diffusible factor, medium from myocytes cultured in 15-ml centrifuge tubes was collected, filtered, and used with an independent low-density myocyte culture. This conditioned medium, however, was unable to prevent the reduction of Kv1.5 mRNA in myocytes cultured at low density (data not shown). In contrast, the loss of Kv1.5 message in live cells was significantly attenuated when myocytes were cocultured with paraformaldehyde-fixed ventricular myocytes (Fig. 3A). Quantitation based on five experiments reveals that myocytes cocultured with paraformaldehyde-fixed myocytes expressed 2.7 ± 0.7-fold more Kv1.5 mRNA than live myocytes cultured alone (Fig. 3B). Kv1.5 mRNA expression in myocytes cultured under low density with wash solution obtained from the fixed myocytes equals that observed in the control cultures (data not shown). It is interesting to note that a number of cell-cell contacts are observed in low-density myocyte cultures, a condition that would lead to ~50% dish coverage in a true cardiac myocyte monolayer. A threefold increase in cell number is required in these dishes to consistently override the mRNA regulation that occurs under low density. This increase in cell number leads to complete coverage of the dish, the formation of multiple layers of cardiac myocytes, and a dramatic increase in the number of cell-cell interactions. Thus Kv1.5 mRNA expression in cultured myocytes parallels that observed in the intact tissue when the magnitude of cell-cell interactions does as well. Furthermore, fixed cells are comparable to live cells in their ability to induce maintained Kv1.5 mRNA levels. Taken together, these findings favor cell contact-dependent signaling in mediation of the effect of cell density on Kv1.5 gene expression.
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To investigate the reversibility of the effect of cell-cell contact, myocytes were cultured at low density for 3 h to downregulate Kv1.5 mRNA expression. Then maximal cell-cell interaction was produced in one group for an additional 3 h. After a total of 6 h in culture, myocytes returned to high-density conditions had more Kv1.5 mRNA than those continuously maintained at low density (Fig. 4). Thus some effect of cell-cell contact is seen after 3 h at low density. However, this recovery is modest compared with the expression seen in continuous high-density cultures. Indeed, the Kv1.5 signal in myocytes incubated at low cell density for 3 h followed by a high cell density incubation for 3 h was only 11 ± 4% of that obtained in myocytes incubated for 6 h at continuously high cell density (n = 4). Thus the downregulation of Kv1.5 mRNA expression observed in low cell-cell contact cultures is essentially irreversible. Yet converting low-density cultures to a high-density setting is able to prevent further loss of Kv1.5 message.
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Alterations in myocyte-myocyte contact also led to changes in Kv4.2 mRNA expression. However, in contrast to Kv1.5, Kv4.2 mRNA expression is inversely proportional to cell-cell contact (Figs. 5A and 6). Thus cardiac myocytes cultured under high density exhibit a time-dependent decrease in Kv4.2 mRNA expression (Fig. 5A). Moreover, Kv4.2 mRNA expression was found to increase when cardiac myocytes were cultured under low-density conditions (Fig. 5A). Figure 5B highlights the contrasting effects of density on Kv1.5 and Kv4.2 mRNA expression in cultured adult cardiac myocytes. When myocytes were cultured at low density for 3 h, there was a sevenfold decrease in Kv1.5 mRNA abundance compared with myocytes cultured at high density for the same length of time. In contrast, these same conditions increased Kv4.2 abundance nearly 2.5-fold. This effect was independent of dexamethasone, since myocytes isolated from naive animals also upregulate Kv4.2 mRNA expression when cultured under low density for 3 h (data not shown).
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The fact that increasing cell density has opposite effects on Kv1.5 and Kv4.2 mRNAs raised the question of whether both genes are affected by the same signal. Paraformaldehyde-fixed myocytes were again employed to determine whether cell-cell contact influenced Kv4.2 mRNA expression. As can be seen in Fig. 6, Kv4.2 mRNA expression in the cocultures approaches those found in high-density cultures and lies well below the levels found in low-density cultures. Quantitation based on three experiments demonstrates that Kv4.2 mRNA expression in live myocytes cocultured with paraformaldehyde-fixed myocytes is 37.1 ± 2.7% of that observed in myocytes cultured under low density alone. Kv4.2 mRNA expression in myocytes cultured under low density with wash solution obtained from the fixed myocytes equals that observed in the control cultures (data not shown). Thus cell-cell contact between cardiac myocytes produces opposite effects on Kv1.5 and Kv4.2 mRNA levels.
If an identical mechanism were regulating both genes, the downregulation of Kv4.2 mRNA, like Kv1.5 mRNA, should be irreversible. To explore this possibility, myocytes were cultured at high density for 3 h to downregulate Kv4.2 mRNA expression. Myocytes from one group were then transferred to 100-mm dishes to decrease the extent of cell-cell contact and cultured for an additional 3 h. Myocytes cultured in this fashion expressed significantly greater amounts of Kv4.2 mRNA than myocytes cultured under high density alone for 3 h (145 ± 8%, n = 3) or 6 h (366 ± 100%, n = 3) (Fig 7). Thus cell contact-mediated regulation of Kv4.2 differs from Kv1.5 in that it is readily reversible.
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DISCUSSION |
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Direct contact between ventricular myocytes influences K+ channel gene expression. This report documents a previously unrecognized stimulus that regulates voltage-gated K+ channel gene expression. We found that Kv1.5 and Kv4.2 mRNA expression in acutely dissociated adult ventricular myocytes is controlled by cell-cell interactions. When myocytes were cultured at low density, a dramatic, rapid, and specific reduction in Kv1.5 message was observed. However, as cell density was increased, Kv1.5 mRNA expression began to reach levels observed in native tissue. In contrast, Kv4.2 mRNA expression was upregulated when myocytes were cultured at low density and downregulated in high-density cultures. In principle, these effect could have been due to the action of a paracrine factor. However, results with conditioned media coupled with coculture experiments using live and paraformaldehyde-fixed cardiac myocytes rule against this possibility. Rather, it appears that direct contact between myocytes is required for maintenance of Kv1.5 and downregulation of Kv4.2 K+ channel gene expression.
The ability of adult cardiac myocytes to influence gene expression via close apposition to another cell type has been previously demonstrated. Primary cultures of adult rat microvascular endothelial cells from ventricular tissue do not express preproendothelin mRNA. However, in the presence of adult rat ventricular myocytes, preproendothelin mRNA levels were upregulated in microvascular endothelial cells (20). To our knowledge, the results presented here represent the first demonstration that adult myocyte-myocyte contacts are important for controlling gene expression.Possible mechanisms of cell contact-dependent control of gene expression. Cardiac myocytes are known to maintain functionally important contacts via intercalated disk structures. However, it is unlikely that those structures underlie the effects of cell-cell contact described here. First, very few end-to-end contacts were observed between myocytes in our cultures. Furthermore, myocytes cultured in pellets could be readily resuspended as individual cells with gentle mixing. This is in contrast to intercalated disks, which form stable connections in vivo. Hence, the effect of cell-cell contact is likely to be mediated by other structures located on the surface of rod-shaped myocytes.
Currently, the identity of these molecules is unknown. However, we have shown that the cell surface signals survive paraformaldehyde fixation. Despite this observation, it seems likely that different pathways are used to regulate Kv1.5 and Kv4.2 expression following changes in cell-cell contact. First, culturing adult cardiac myocytes at low density leads to reciprocal effects on Kv1.5 and Kv4.2 mRNA expression. Second, although fixation does not destroy the signal, incubating cells at low density results in an attenuation of the cell density effect on Kv1.5 mRNA expression. This raises the possibility that for Kv1.5 either the cell surface molecules involved in cell-cell contact or a signaling pathway required by these cell surface molecules is labile in live cells. In contrast, cell density control of Kv4.2 mRNA expression is stable. Thus myocyte-myocyte contact might affect multiple signaling mechanisms. It is possible that cell-cell contact regulates cardiac Kv1.5 and Kv4.2 gene transcription. Studies with nonchannel genes in other tissues have shown that density-dependent upregulation of gene expression is associated with elevated transcription rates (3, 4, 33). In addition, two groups have identified promoter elements and nuclear factors responsible for conferring sensitivity to changes in the extent of cell-cell contact (11, 18). Because the half-life for Kv1.5 mRNA has been estimated to be ~30 min in clonal rat pituitary cells (27), specific attenuation of gene transcription under low contact settings could explain the rapid downregulation of Kv1.5 channel mRNA. If this hypothesis is correct, our results argue that the half-life for Kv4.2 mRNA is also short (i.e., <2 h). Alternatively, changes in cell-cell contact could regulate Kv1.5 and Kv4.2 mRNA stability. Further work will be required to distinguish between these possibilities.Significance of cell contact-dependent K+ channel gene expression. The fact that K+ channel gene expression can change dramatically with cell density has three implications. First, this finding is pertinent to studies aimed at determining the molecular basis for the cardiac K+ currents. Some groups have approached this goal by examining the effects of antisense oligonucleotides (7, 8) or dominant negative subunits (12) on K+ currents in cultured cardiac myocytes. Others have employed animal models to correlate changes in K+ channel gene expression with voltage-gated K+ currents (10, 21, 23, 32, 28). The relevance of such studies rests on the assumption that the assortment of channel genes expressed in vitro represents the in vivo situation. Our results illustrate that this assumption should be verified before researchers embark on such studies.
Second, cell contact-dependent regulation of cardiac K+ channel gene expression may be important under pathological conditions. It is known that significant ventricular remodeling occurs in necrotic and adjacent regions following myocardial infarctions. Several laboratories have found reduced cell-cell contact in ischemic heart disease (16, 24, 31). Our results raise the possibility that changes in cell contact-dependent signaling produced by these conditions may influence excitability by altering expression of voltage-gated channels. Finally, because the effects of cell-cell contact have not been previously examined in adult cardiac myocytes, it is possible that the effects on Kv1.5 and Kv4.2 messages are not unique. It is already known from studies in brain that cell-cell contact regulates astrocytic basic fibroblast growth factor gene expression in response to injury or neuronal degeneration (9, 15). Applying the approaches described here may establish similar contact-dependent control of growth factor genes in the heart. Furthermore, because the effects on channel mRNAs develop rapidly, it is also possible that cell contact-dependent signaling may produce acute effects on a variety of targets (e.g., channel and contractile proteins). Thus the sensitivity of K+ channel genes to a wide variety of signals (14) may have facilitated the discovery of a significant general regulatory mechanism in the heart. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Koichi Takimoto for comments.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-55312 and a Grant-in-Aid from the American Heart Association (Pennsylvania Affiliate). K. M. Hershman was supported by individual NHLBI Postdoctoral Fellowship Grant HL-09347.
E. S. Levitan is an Established Investigator of the American Heart Association.
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. §1734 solely to indicate this fact.
Address for reprint requests: E. S. Levitan, E1351 Biomedical Science Tower, Dept. of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261.
Received 1 April 1998; accepted in final form 20 August 1998.
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