© 1995 American Heart Association, Inc.
Articles |
Expression of Multiple Connexins in Cultured Neonatal Rat Ventricular Myocytes
From the Departments of Pediatrics, Cell Biology and Physiology, Medicine, and Pathology, Washington University School of Medicine, St Louis, Mo, and the Department of Genetics and Cell Biology (P.D.L.), University of Minnesota, St Paul.
Correspondence to Eric C. Beyer, MD, PhD, Department of Pediatrics, Box 8116, Washington University School of Medicine, One Children's Place, St Louis, MO 63110.
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Abstract |
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Abstract Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from [35S]methionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with [32P]orthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.
Key Words: gap junctions • intercellular communication • electrical conduction • ion channels • phosphorylation
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Introduction |
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Electrical conduction in the heart is dependent on current transfer at gap junctions, specialized appositional membrane regions containing densely packed intercellular channels.1 Electrophysiological observations have implicated spatial rearrangements of gap junctions as a critical determinant of the slow conduction and block leading to reentrant arrhythmias.2 3 Morphometric studies have demonstrated remodeling of gap junctions in infarct border zones, which are a critical site for arrhythmogenesis.4 Evaluation of the molecular controls of cardiac gap junction expression, assembly, and modification is crucial to understanding these processes.
Gap junction channels are multimeric assemblies of subunit proteins called connexins (reviewed by Beyer5 ). Each connexin has a similar predicted topological structure, with four conserved transmembrane domains, two highly conserved extracellular regions, and two unique cytoplasmic regions. Each connexin forms channels with distinct regulatory and conductance properties6 ; these physiological differences likely derive from the unique portions of their sequences.
Three different connexins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40).7 8 DNAs encoding these connexins have been cloned from multiple species.7 9 10 11 12 13 14 The predicted connexin protein sequences have many similarities, and each sequence has several consensus sites for phosphorylation, which may be important in the regulation of gap junction channels (reviewed by Saez et al15 ). Although Cx43, Cx45, and Cx40 are all expressed in canine ventricular myocytes, their relative abundances vary in different regions of the heart with different conductive properties.16 17 18 19 20 21 Expression of Cx43, Cx45, and Cx40 by stable transfection of a communication-deficient cell line has shown that each connexin forms channels with unique electrical properties.6 22
To begin to elucidate the determinants of cardiac intercellular
coupling and the mechanisms mediating remodeling of gap junctions in
diseased myocardium, the present study was undertaken to examine
the expression, turnover, and modification of Cx43, Cx45, and Cx40 in
cultured neonatal rat ventricular myocytes. Primary cultures of
neonatal rat ventricular myocytes have become a widely used model
system for in vitro investigation of the regulation of cardiac gene
expression and cardiac protein biosynthesis and
phosphorylation.23 24 25 Previous studies of Cx43 expression
and biosynthesis in cultured neonatal rat ventricular myocytes have
identified Cx43 as a phosphorylated protein with a half-life of 
2
hours.26 27 28
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Materials and Methods |
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All reagents were obtained from Sigma Chemical Co unless otherwise indicated.
Isolation and Culture of Neonatal Rat Ventricular Myocytes
Primary cultures of neonatal rat ventricular myocytes were
prepared according to the procedure described by Engelmann et
al25 with minor variations. The hearts were removed from
anesthetized 1-day-old Sprague-Dawley rats (Sasco, O'Fallon,
Mo) and trimmed of atrial tissue and great vessels. The ventricles were
rinsed, minced on ice, and digested with 125 U/mL type II collagenase
(Worthington Biochemicals) in Joklik's modified Eagle's medium
(GIBCO) supplemented with 100 U/mL penicillin, 100 µg/mL
streptomycin, 250 ng/mL amphotericin, and 50 µmol/L CaCl2
at 37°C. Cells were harvested after digestion and resuspended in
ice-cold collagenase-free digestion medium containing 0.5% fetal
bovine serum. Pooled cells were passed through a 70-µm nylon filter,
layered over a Percoll cushion (69.6% Percoll in phosphate-buffered
saline [PBS]; specific density, 1.095), and centrifuged at
1700g for 10 minutes. Cells were resuspended in PC-1 medium
(Hycor Biochemical) supplemented with penicillin, streptomycin, and 0.1
mmol/L bromodeoxyuridine. The cells were plated at 500 to 750
myocytes per square millimeter on 60-mm culture dishes coated with
either collagen or an extracellular matrix from cultured NRK cells.
Cultures were rinsed the following morning and replenished with a 1:2
mixture of PC-1 and Hams F-12/DMEM (GIBCO) with penicillin,
streptomycin, amphotericin, and bromodeoxyuridine at concentrations
previously indicated; medium was replenished daily. Cell cultures were
maintained for 72 hours before RNA preparation or
immunoprecipitation, by which time the myocytes had formed a
synchronously contracting syncytium. At the time of harvest, we
estimated that >95% of living cultured cells were myocytes.
RNA Isolation and Blotting
Total cellular RNA was prepared from cultured neonatal rat
ventricular myocytes as described by Chomczynski and
Sacchi,29 separated on a 1% agarose/formaldehyde gel, and
transferred overnight to nylon membranes as previously
described.30 Relative equivalence of loading of samples
and RNA integrity were verified by ethidium bromide staining and by
hybridization of blots with a probe for 18S rRNA. Specific probes were
generated from rat Cx43, dog Cx45, and rat Cx40 DNAs by random primer
labeling (Boehringer Mannheim) with [32P]dATP (New
England Nuclear) and hybridized to blots as previously
described.7 11 The probes were hybridized under conditions
previously determined to detect specific connexin transcripts.
Anti-Connexin Antibodies
A rabbit antiserum directed against a synthetic peptide
representing amino acids 252 to 271 in rat Cx43 was produced
previously and has been extensively characterized.31 A
mouse monoclonal antibody (IgG1) against Cx43 (amino acids 252 to 270)
was purchased from Zymed. Rabbit polyclonal antisera directed against
synthetic peptides representing amino acids 285 to 298 of
canine Cx45 and amino acids 316 to 329 of canine Cx40 were purified by
chromatography on agarose derived with the specific immunogenic
peptides as described previously.8 The specificity of
these antibodies has been demonstrated by immunofluorescence and by
immunoprecipitation of in vitro–translated connexins.8
Some experiments also used an antiserum raised against residues 260 to
279 of chick connexin42 (Cx42), which exhibits reactivity with
mammalian Cx40.7 20
Immunofluorescent Labeling of Cultured Cells
Cells were cultured in plastic chamber microscope slides (Nunc),
fixed in 50% methanol/50% acetone for 2 minutes at room temperature,
and permeabilized in 1% Triton X-100/PBS for 10 minutes at room
temperature. Cells were incubated in primary antibodies (mouse
monoclonal anti-Cx43 or affinity-purified rabbit polyclonal anti-Cx45
or anti-Cx40) at 1:200 dilution overnight at 4°C, washed extensively,
and then incubated with secondary antibodies (Texas red–conjugated
goat anti-rabbit IgG or fluorescein-conjugated goat anti-mouse IgG)
(Jackson ImmunoLabs) at 1:800 dilution for 3 hours. The cells were
examined with a Zeiss epifluorescence microscope or with an MRC-500
laser scanning confocal microscope (Bio-Rad).
Immunoprecipitation and Determination of Protein Turnover
Dynamics
Immunoprecipitation of radiolabeled connexin proteins was
performed according to the methods of Laing et al.32 Cells
were labeled in methionine-depleted medium (50% DMEM/50% F-12 medium)
containing [35S]methionine (100 µCi/mL, Amersham) or in
phosphate-depleted medium containing [32P]orthophosphate
(100 µCi/mL, New England Nuclear). For pulse-chase experiments, after
labeling for 2 hours with [35S]methionine, cells were
rinsed in PBS and incubated in normal culture medium for the designated
chase time. The cells were rinsed and scraped in PBS and lysed by
sonication (four times for 15 seconds). Cellular debris was
concentrated by centrifugation (10 minutes, 14 000g), and
connexins were solubilized by boiling the pellet in RIPA buffer (PBS
containing 1% Triton X-100, 0.6% sodium dodecyl sulfate [SDS], 100
U/mL aprotinin [Boehringer Mannheim], 0.1% phenylmethylsulfonyl
fluoride, and 1 mmol/L sodium orthovanadate) for 5 minutes. After
centrifugation (10 minutes, 14 000g), the supernatant was
incubated with 20 µL rProtein A-IPA 300 (Repligen) and 5 to 10 µL
of the specific antibodies, with shaking at 4°C for 2 hours. Pellets
were collected with a brief centrifugation, washed overnight and then
three times for 30 minutes in RIPA buffer at 4°C, analyzed by
SDS–polyacrylamide gel electrophoresis (PAGE) on a 12.5% gel, and
subjected to fluorography after treatment with ENH3ANCE
(New England Nuclear). Densitometric images were generated by using a
Dage-MTI CCD72 camera (Dage) and digitized with a Matrox MVP
image-processing board. Gray-scale values of polypeptide bands were
quantified by using an FL-4000 (Georgia Instruments). The relative
amount of each connexin protein was determined by dividing the
densitometric value by the product of the number of methionines in the
polypeptide (7 for Cx43, 11 for Cx45) and the percentage of the total
protein pool synthesized during a 2-hour incubation (estimated by using
the half-life for each protein as determined below). The first-order
decay constant (k) was calculated from best-fit single exponential
decay curves of the form y=e(-kt) generated with the
program ENZFITTER (Elsevier Biochemical). The half-life
of each protein was determined according to the formula
t1/2=0.693/k.
Phosphoamino Acid Analysis
Cx45 was immunoprecipitated from cells labeled with
[32P]orthophosphate for 6 hours in phosphate-depleted
medium (50% DMEM/50% F-12 medium buffered with HEPES) as outlined
above. After SDS-PAGE, the gel was blotted onto Immobilon-P membranes
(Millipore) by using a semidry transfer apparatus (Bio-Rad). The blot
was exposed to x-ray film to determine the location of the Cx45 band.
Cx45 was excised from the membrane and hydrolyzed in 5.7N HCl as
described previously,33 except the membrane was hydrolyzed
for 1 hour at 105°C in a glass ampule sealed under nitrogen. The acid
was transferred to another tube, and the membrane was washed once with
water. The acid and wash were combined and concentrated in a Speed-Vac,
dissolved in 100 µL water, and reconcentrated. Nonradioactive
phosphoamino acids (2 µg each per lane) were added to the samples,
and electrophoresis of the phosphoamino acids was performed at pH 3.5
(H2O/acetic acid/pyridine, 945:50:5) on Whatman No. 3 paper
at 750 V as described previously.34 Control phosphoamino
acid spots were detected by spraying with ninhydrin. Autoradiography
was performed with Kodak XAR film with a Dupont Cronex intensifying
screen.
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Results |
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Connexin mRNA Expression by Cultured Neonatal Rat Ventricular Myocytes
To determine whether cultured neonatal rat ventricular myocytes expressed the connexin mRNAs previously identified in adult mammalian cardiac myocytes, we hybridized total cellular RNA isolated from 72-hour-old neonatal rat ventricular myocyte cultures with radiolabeled DNA probes specific for Cx43, Cx45, and Cx40. As shown in Fig 1
, all three probes hybridized with RNA from the
cultured cells. Cx43 and Cx40 probes identified transcripts of 3.4
kb as previously described in rat heart and cultured rat cell
lines.11 30 The Cx45 probe hybridized to a band of 2.1
kb, as seen previously in dog and mouse heart.7 14 Samples
of myocyte RNA also hybridized with probes containing specific 3'
coding or noncoding sequences from the rat Cx40 cDNA, confirming the
presence of Cx40 mRNA in the cultured myocytes (data not shown).
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Immunolocalization of Connexins in Neonatal Rat Ventricular
Myocytes
Immunofluorescent staining was performed on permeabilized cultured
rat ventricular myocytes. Anti-Cx43 and anti-Cx45 antibodies both
produced similar staining patterns in these cells, whereas no
immuoreactive sites were detected with anti-Cx40 antibodies. Panels A
and B of Fig 2 show a representative field from
cells double-labeled with the mouse anti-Cx43 and rabbit anti-Cx45
antibodies and viewed by epifluorescent light microscopy. Panel C shows
the same field viewed under bright-field conditions. The pattern of
staining with the two antibodies is very similar. Panels D and E show a
confocal microscope image from a different myocyte preparation. Both
the anti-Cx43 and anti-Cx45 antibodies produced punctate labeling along
appositional membranes between cells. The distribution of
immunoreactive sites appeared identical by computer superposition. A
low level of cytoplasmic staining was seen with both antibodies. When
this experiment was repeated with the mouse monoclonal anti-Cx43 and
rabbit anti-Cx40 antibodies (panels F and G), only Cx43
immunoreactivity was detected in the myocyte membranes.
Immunofluorescent staining of frozen sections of neonatal rat ventricle
gave similar results: Cx43 and Cx45 were present between
ventricular myocytes, but no immunoreactive Cx40 was detected (data not
shown). Control experiments using secondary antibodies alone or using a
primary antibody followed by the noncorresponding secondary reagent
(eg, rabbit primary antibodies followed by fluorescent goat anti-mouse
IgG) yielded no significant labeling; also, no significant
immunoreactivity was detected either in cultured cells or in intact
tissue samples when using an antiserum raised against a chick Cx42
peptide sequence, which cross-reacts with the mammalian Cx40
protein7 20 (data not shown).
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Immunoprecipitation of Connexins From Metabolically Labeled
Myocytes
Parallel cultures of neonatal rat ventricular myocytes were
incubated for 2 hours with [35S]methionine,
immunoprecipitated with anti-Cx43, anti-Cx45, or anti-Cx40 antibodies,
and analyzed by SDS-PAGE and fluorography. As shown in Fig 3, Cx45 and Cx43 polypeptides were separately isolated
from the cell lysates. The Cx43 immunoprecipitate contained three
distinguishable bands that migrated at 42, 44, and 45 kD. A similar
pattern has been reported by other laboratories.26 35 36
The anti-Cx45 antiserum immunoprecipitated a predominant 48-kD band,
which was accompanied by a 46-kD polypeptide in some experiments. We
have immunoprecipitated polypeptides of the same size from several cell
lines that express Cx45 mRNA32 37 ; incubation in the
presence of the cognate Cx45 peptide blocked the precipitation of both
bands. The 46-kD band likely represented a degradation
product occurring during protein isolation, since it was inconsistently
observed, it turned over with the same kinetics as the 48-kD band, it
was blocked by peptide, and it had a faster mobility than Cx45
translated in vitro.32
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The relative amounts of Cx43 and Cx45 proteins were estimated by
immunoprecipitating parallel extracts from individual cultures. Cx43
and Cx45 were present in comparable amounts in such experiments
(Fig 3
), and analysis of multiple experiments consistently showed
that Cx45 accounted for 50% to 75% of the precipitated connexin
polypeptides. Reprecipitation with the same anti-connexin antibodies
from the supernatants of previously immunoprecipitated samples yielded
no radiolabeled proteins, suggesting that our procedure was
quantitative. No radiolabeled polypeptides were specifically
precipitated from [35S]methionine-labeled myocyte lysates
by anti-Cx40 or anti-Cx42 antibodies (data not shown).
Phosphoamino Acid Analysis of Cx43 and Cx45
To examine phosphorylation of the connexins in the cultured
neonatal myocytes, Cx43 and Cx45 were immunoprecipitated from cultures
that had been labeled with either [32P]orthophosphate or
[35S]methionine (Fig 4A). The anti-Cx45
antibodies precipitated a polypeptide of 48 kD from both the
[35S]methionine- and
[32P]orthophosphate-labeled cells. The anti-Cx43
antibodies precipitated 42- and 44-kD polypeptides labeled with
[35S]methionine and a broad band of 44 kD from the
[32P]orthophosphate-labeled myocyte lysate. Phosphoamino
acid analysis was performed on 32P-labeled Cx45 and
Cx43. Only phosphoserine was detected in Cx45 (Fig 4B).
32P-labeled Cx43 contained both phosphoserine (85%) and
phosphothreonine (15%) residues (data not shown), consistent with
observations from other laboratories.27 35 38 39
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In Vitro Half-Lives of Cx45 and Cx43
We determined the half-lives of both Cx45 and Cx43 proteins in our
cultures by using the pulse-chase method. After a 2-hour incubation
with [35S]methionine, cultures were rinsed with PBS and
incubated with normal medium for selected intervals of 0 to 8 hours.
Cx45 was immunoprecipitated from each culture and resolved by SDS-PAGE
and fluorography. As shown in Fig 5 (top), the specific
activity of Cx45 decreased markedly during an 8-hour chase in normal
medium. The maximum incorporation of [35S]methionine
occurred within the first hour of the chase. Densitometric
quantification and exponential curve fitting of the data from multiple
experiments were used to determine the half-lives of Cx45 and Cx43 (Fig 5 [middle and bottom]). The half-lives of Cx45 and Cx43 proteins were
calculated from best-fit single-exponential decay curves. The decay
constant for Cx45 was 0.24±0.025 h-1, which
corresponded to a half-life of 2.9 hours (range, 2.6 to 3.2 hours). The
Cx43 data were best fit by a curve with a decay constant of 0.37±0.055
h-1, which corresponded to a half-life of 1.9 hours
(range, 1.6 to 2.2 hours).
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Discussion |
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The results of the present study show that neonatal rat ventricular myocytes express the mRNAs encoding three connexins (Cx43, Cx45, and Cx40). These three connexin mRNAs have been detected previously in adult canine ventricular myocytes,7 in the hearts of animals of various species,7 11 13 14 40 and in fetal human hearts.41 This suggests that these cultured cells may be an appropriate model for studying the regulation of cardiac gap junction expression, modification, and remodeling. Although other laboratories have reported that the expression of some cardiac genes, including Cx43, varies with the density of the plated cells or the amount of time in culture,42 43 44 we have attempted to standardize these variables in our experiments.
Neonatal rat ventricular myocytes also expressed two connexin proteins (Cx43 and Cx45), as detected by both immunofluorescence and immunoblotting. The failure to detect Cx40 protein suggests that this protein may be regulated in a manner different from the other connexins, perhaps by translational mechanisms; ie, Cx40 mRNA is transcribed but the protein is either not translated or is translated and then rapidly degraded. However, we cannot absolutely rule out the presence of Cx40 protein, because there are potential alternative explanations for the lack of Cx40 detection that are difficult to exclude: (1) Whereas Cx40 protein is detected by immunofluorescence in sections of canine,16 human,40 or rat heart (authors' unpublished data) and our antibodies will immunoprecipitate in vitro–translated rat Cx40,8 it is possible that the protein was posttranslationally modified in a manner that masked the epitope recognized by our Cx40 antibodies. (2) If Cx40 had a very long half-life (much longer than Cx43 or Cx45), it would not be labeled sufficiently during the 2-hour incubation in [35S]methionine. (3) If Cx40 was not assembled into cell surface gap junctional plaques in these cells, it would not be detected by immunofluorescence. We tried to test these possibilities by repeating the experiments by use of a second antiserum reactive with Cx40 (anti-chick Cx42c, amino acids 260 to 279), which also did not detect Cx40 protein (data not shown).
In fact, although surprising, our data on Cx40 expression in neonatal rat ventricular myocytes are consistent with most previously reported observations regarding Cx40 protein expression in ventricular myocardium in vivo. The majority of studies from our laboratory and others have found little immunoreactive Cx40 protein in ventricular cardiac myocytes in vivo.16 17 18 19 20 21 41 No studies using in situ hybridization to localize Cx40 mRNA expression in rat ventricle have been published. Immunoreactive Cx40 is enriched in the atrium and nodes and bundles of the conducting system and vascular endothelium.16 17 18 19 20 21 Although abundant Cx40 mRNA has been detected in heart or ventricular homogenates,7 12 13 18 only limited in situ hybridization studies of this tissue have been conducted, so it is not possible to explain the cellular source of this Cx40 mRNA.
Our data have demonstrated several similarities in the expression and modification of Cx45 and Cx43 proteins. Both proteins are abundant in these cells, they are phosphoproteins, they colocalize at distinct spots within appositional regions of the myocyte membranes, and they have relatively short half-lives for membrane proteins. Colocalization of multiple connexins within the same gap junction plaques has previously been demonstrated in cardiac myocytes and other cells.8 45
Although Cx43 and Cx45 are both phosphorylated, several lines of biochemical data suggest differences in the sites and manner of phosphorylation. Cx43 is phosphorylated on both serine and threonine residues,26 27 39 46 whereas only phosphoserine was detected in Cx45. Phosphorylation of Cx43 results in production of multiple forms of the protein, as detected by SDS-PAGE, that have reduced electrophoretic mobility. Several investigators have demonstrated that each of these bands contains multiple phosphorylated forms of Cx43.39 46 47 In contrast, phosphorylation of Cx45 results in no change in electrophoretic mobility. These results suggest substantially more heterogeneity of phosphorylation of Cx43 than has been observed for Cx45.
The kinases involved in phosphorylation of Cx43 or Cx45 have not been determined. However, several connexins (including Cx43 and Cx45) contain putative phosphorylation sites for protein kinase C (and other kinases) within carboxyl-terminal regions.7 30 48 Synthetic peptides corresponding to some of these sequences in Cx43 can act as substrates for protein kinase C in vitro.49 Phosphorylating and dephosphorylating treatments affect both macroscopic junctional currents and single-channel behavior in neonatal rat ventricular myocytes.50 51 52 Cx43 phosphorylation also appears to affect the incorporation of connexins into gap junctional plaques.36 It is possible that modulation of connexins by cellular protein kinases may have different effects on different connexins. Indeed, treatment of the BWEM cell line with a phorbol ester increased phosphorylation of Cx43 but decreased synthesis and phosphorylation of Cx45.32
Using pulse chase studies, we demonstrated that Cx45 turns over with a
half-life of 3 hours. In our studies, Cx43 also had a short
half-life (2 hours), similar to that previously observed in cardiac
myocytes26 and other cells.35 53 The best-fit
exponential functions fit the data over the entire decay phase of the
chase, and the calculated half-lives are not significantly altered if
only the first 6 hours of the chase are considered. Many, but not all,
connexins have short half-lives as detected in vivo and in vitro;
although the liver gap junction proteins Cx32 and Cx26 have half-lives
comparable to those reported here for Cx43 and Cx45, the lens gap
junction protein Cx46 has an apparent half-life of >1
day.42 54 55 56 Compared with other membrane proteins, these
gap junction proteins exhibit rapid turnover. These findings suggest
that synthesis and degradation of gap junctional channels is a very
dynamic process and may be a major mechanism for the regulation of
cardiac intercellular coupling and potential remodeling of myocardial
cellular connections. Agents that cause minor perturbations of Cx43 or
Cx45 turnover might thus have significant consequences regarding the
relative and total amounts of connexin proteins present in
myocytes.
The determinants of turnover of connexins are currently unknown. Some proteins that have short half-lives contain a sequence rich in proline, glutamic acid, serine, and threonine residues, called a PEST sequence.57 As discussed by Laird et al,26 potential PEST sequences are present in the Cx43 protein. One PEST-like region is also found in Cx45 (amino acids 135 to 149). The importance of these sequences in control of connexin degradation merits further investigation.
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-45466, HL-07275, GM-46277, and HL-17646 SCOR in Coronary Vascular Disease. Dr Laing was supported by a fellowship from the Lucille P. Markey Foundation. Dr Beyer was supported by an Established Investigator Award from the American Heart Association. We thank Karen Green and Eileen Westphale for invaluable technical assistance. We also thank Dr Dorothy Schafer for assistance in the use of the confocal microscope.
Received March 22, 1994; accepted November 14, 1994.
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