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J Biol Chem, Vol. 273, Issue 48, 32158-32166, November 27, 1998
From the Department of Pharmacology and Physiology, University of
Rochester, School of Medicine and Dentistry,
Rochester, New York 14642
We have investigated the mechanisms by which
stimulation of cardiac muscarinic receptors result in paradoxical
stimulatory effects on cardiac function, using cultured neonatal rat
ventricular myocytes as a model system. Application of low
concentrations of carbachol (CCh) (EC50 = 35 nM) produced an atropine-sensitive decrease in spontaneous
contraction rate, while, in cells pretreated with pertussis toxin,
higher concentrations of CCh (EC50 = 26 µM)
elicited an atropine-sensitive increase in contraction rate. Oxotremorine, an m2 muscarinic acetylcholine receptor
(mAChR) agonist, mimicked the negative but not the positive
chronotropic response to CCh. Reverse transcription followed by
polymerase chain reaction carried out on mRNA obtained from single
cells indicated that ventricular myocytes express mRNA for the
m1, m2, and, possibly, m4 mAChRs.
The presence of m1 and m2 mAChR protein on the
surface membranes of the cultured ventricular myocytes was confirmed by
immunofluorescence. The CCh-induced positive chronotropic response was
significantly inhibited by fluorescein-tagged antisense
oligonucleotides directed against the m1, but not the m2 and m4, mAChR subtypes. The response was
also inhibited by antisense oligonucleotides against Gq Stimulation of postsynaptic muscarinic acetylcholine receptors
(mAChRs)1 on cardiac cells is
able to produce dual effects on the heartbeat. Classically, stimulation
of the vagus nerve or the application of low concentrations of
muscarinic cholinergic agonists produces characteristic negative
chronotropic and inotropic effects on cardiac tissue and cells (1).
Paradoxically, under appropriate conditions, activation of cardiac
mAChRs elicits stimulatory effects on the rate of beating and
contractile force of the heart. These latter effects often require
higher concentrations of agonist and in some cardiac cell types are
only seen after pretreatment with pertussis toxin (PTx) (2-4).
It has been widely believed, based on both radioligand binding
techniques as well as Northern blot analyses, that cardiac cells
express only the m2 mAChR subtype on their surface
membranes (5-8). However, more recent findings suggest limits in the
sensitivity of these techniques in detecting low levels of protein and
mRNA (6). For example, it has been demonstrated that mRNA for
mAChR subtypes that were missed by Northern blot studies were detected by use of reverse transcription followed by polymerase chain reaction (RT-PCR) in neuroblastoma cells as well as in guinea pig ventricular myocytes (7, 9). Previous work in this laboratory, employing the
techniques of single-cell RT-PCR on cardiac myocytes isolated from
adult rat ventricle has demonstrated the existence of m1 mAChR mRNA in addition to m2 mRNA (10, 11).
Furthermore, we have demonstrated that the activation of m1
mAChRs leads to an increase in muscle shortening and Ca2+
transients. However, the signal transduction pathway mediating this
stimulatory response is still unknown. Moreover, there is no report on
the role of m1 mAChRs in the regulation of heart rate.
In the present studies, we have taken advantage of the ability of
cultured neonatal rat ventricular myocytes to contract spontaneously and have utilized them as a cardiac cell model system to investigate the role of m1 mAChRs in the regulation of rate of
contraction. By applying a number of complementary approaches,
including single-cell intracellular Ca2+ concentration
([Ca2+]i) measurements, single-cell RT-PCR,
immunocytochemistry and antisense technology, four specific questions
were addressed. 1) Do cultured neonatal rat ventricular myocytes
exhibit dual responses to mAChR stimulation in a manner similar to that
observed in other cardiac cell preparations? 2) How many subtypes of
mAChRs are expressed in cultured neonatal rat ventricular myocytes? 3) Which mAChR subtype mediates the stimulatory effects of muscarinic agonists on the rate of contraction? 4) What second messenger system is
involved in generating this response?
A preliminary report of this work has appeared in abstract form
(12).
Culture of Ventricular Myocytes--
Cultured neonatal rat
ventricular myocytes were prepared using modifications of a previously
published procedure (13). Briefly, 1-3-day-old Sprague-Dawley rats
were anesthetized with ether and decapitated, and their hearts
aseptically excised. The bottom half of the hearts were cut, rinsed in
magnesium- and calcium-free phosphate-buffered saline (PBS), and finely
minced with a pair of scissors. Single cells were obtained by brief
alternating periods of enzymatic digestion with 0.125% trypsin and
mechanical disaggregation at room temperature. The cells dispersed in
the supernatant from the first two digestions were discarded. The
supernatant from the following digestions were collected in growth
medium consisting of Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 50 units/ml
penicillin/streptomycin, 10% calf serum, and 10% horse serum. When
all the tissue was digested, the cells were pelleted by centrifugation
at 200 × g in a tabletop centrifuge. The pellet was
resuspended in growth medium and the cells preplated in 10-cm diameter
tissue culture dishes. After 2-3 h, unattached cells were plated on
25-mm diameter, circular glass coverslips in 35-mm tissue culture wells
(~1 × 104 cells/well). The cultures were exposed to
3500 rads of Measurements of [Ca2+]i in Single
Ventricular Myocytes--
The method for recording of Ca2+
transients from single cardiac myocytes has been published previously
(10, 15). Ventricular myocytes cultured on glass coverslips were loaded
with the membrane-permeable acetoxymethyl ester form of fura-2 (200 nM fura-2 AM for 10 min) at room temperature in HEPES
buffer. The buffer solution contained (mM): NaCl 145, KCl
5, CaCl2 2, MgCl2 1, glucose 10, HEPES 10, and
1 mg/ml bovine serum albumin (pH 7.4). After incubation, cells were
allowed to sit for >30 min to allow the complete conversion of fura-2
AM to fura-2 free acid.
For measurements of [Ca2+]i transients,
coverslips with dye-loaded cultured ventricular myocytes were mounted
in a tissue chamber on the stage of a Nikon Diaphot inverted microscope equipped for epifluorescence. Single cells were alternately excited at
340 and 380 nm wavelength light from a Xenon-arc UV lamp through a 40×
oil immersion objective. The emitted signal at 510 nm was focused onto
the photocathode of a photon-counting photomultiplier tube. The signals
in response to the two excitation wavelengths were digitized and stored
in a microcomputer. All experiments were conducted at 37 °C with
continuous perfusion of cells with HEPES buffer containing no drugs
(control recordings) or the indicated concentrations of drugs in a
final constant bath volume of 1 ml.
Single-cell Reverse Transcription-Polymerase Chain
Reaction--
The method of single-cell RT-PCR was adapted from
previously published protocols (10, 16). Briefly, glass micropipettes of the type used for conventional patch clamp studies were fashioned and back-filled with approximately 3 µl of RNase-free water. The micropipettes were used to suck up the contents of single cultured ventricular myocytes identified in culture by the application of a
gentle suction. Entry of the cellular contents into the micropipette was monitored under the light microscope. The aspirated cell contents were then extruded into an Eppendorf tube that contained a reverse transcription master mix. The master mix contained: 2 µl of 10× RT
buffer (100 mM Tris-HCl, 500 mM KCl, 1% Triton
X-100), 10 mM MgCl2, 0.5 mM each
dNTP, 10 mM dithiothreitol, 20 units of RNase inhibitor,
and 0.5 µg of oligo(dT) primer. Since the intracellular contents
harvested included the nucleus, 1 unit of RNase-free DNase was added to
the mixture to remove genomic DNA. Following a 30-min incubation at
37 °C, the DNase was inactivated by heating the sample at 95 °C
for 5 min and then placing it on ice. Reverse transcription was
initiated by the addition of 200 units of Superscript II reverse
transcriptase (Life Technologies, Inc.) followed by incubation of the
sample at 42 °C for 1 h. For negative controls, water was added
instead of reverse transcriptase. For the PCR reactions, 4-10 µl of
the RT product was added to a reaction solution (50 µl final volume)
consisting of: 2-5 mM MgCl2, 5 µl 10× PCR buffer (100 mM Tris-HCl, 500 mM KCl), 0.2 mM each dNTP, 50 pmol of each PCR primer, and 2.5 units of
AmpliTaq DNA polymerase (Perkin Elmer). The reaction mixture was
overlaid with mineral oil and the PCR amplifications carried out in an
automated thermocycler for 45-50 cycles (94 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min). Ten µl of the PCR products were
electrophoresed on 1.2% agarose gels containing 0.5 µg/ml ethidium
bromide and photographed under UV illumination.
The primer sequences used for the PCR reactions and the positions
of the published rat mAChR cDNAs that they corresponded to (17-20)
are as follows.
Immunofluorescence Detection of mAChR Subtypes--
Subtypes of
mAChR protein expressed on the surface membranes of cultured neonatal
rat ventricular myocytes were probed by immunofluorescence as described
previously (10). Briefly, cultured ventricular myocytes were fixed with
3% paraformaldehyde for 10 min and subsequently permeabilized with PBS
containing 0.3% Triton X-100. Nonspecific binding sites were blocked
by preincubating the fixed cells for 30 min with PBS containing 10%
normal goat serum (GPBS). Cells were then exposed to GPBS, containing
either no primary antibody or affinity-purified rabbit polyclonal
antibodies specific for the m1 and m2 mAChRs
(diluted 1:200; 0.5 µg/ml final concentration) for 48 h at
4 °C. These antibodies have been characterized by
immunoprecipitation and immunocytochemistry using the family of cloned
mAChRs as well as receptors expressed in their native tissues (20). At
the end of the incubation period, excess primary antibody was removed
by washing with GPBS and the cells were exposed to a
fluorescein-labeled goat anti-rabbit secondary antibody, used at 1:500
dilution, for 45 min at room temperature. Following the removal of
excess secondary antibody by washing with PBS, the cells were mounted
on glass slides in Mowiol 4-88 (Calbiochem). Images were acquired
through the use of a confocal microscope.
Functional Inhibition of Gene Expression with Antisense
Oligonucleotides: Design and Fluorescein Labeling of
Oligonucleotides--
Antisense oligonucleotides targeted to mRNA
for the m1, m2, and m4 mAChRs as
well as to Gq
The modified oligonucleotides were labeled with fluorescein
using established methods (24). Briefly, 0.1 µmol of oligonucleotide was incubated with 0.6 µmol of fluorescein isothiocyanate for 18 h in 1 ml of sodium carbonate buffer (pH 9). The reactions were
terminated by adding 50 µl of 100 mM ammonium chloride,
and after another 2 h the fluorescein-tagged oligonucleotide was
separated from free fluorescein isothiocyanate by spun column
chromatography using Sephadex G-25 equilibrated with deionized water.
The labeled oligonucleotides were sterilized by filtration through a
0.2-µm membrane filter and quantitated by measuring the absorbance at 260 nm (A260). One A260
unit was taken as being equivalent to 30 µg of oligonucleotide.
Treatment of Cells with Oligonucleotides--
Antisense
oligonucleotides were dispensed from a stock concentration of 100 µM stored at Nuclear Localization of Oligonucleotides--
To identify cells
with nuclear localization of fluorescein-tagged oligonucleotides, they
were viewed by fluorescence microscopy. Cells were placed on the stage
of a Nikon Diaphot inverted microscope equipped for epifluorescence.
Selected cells were excited with 490 nm wavelength light through a 40×
oil immersion objective. The emitted fluorescence images were acquired
at 530 nm with a dichroic mirror focused onto a SIT camera.
Intracellular organelles such as the nucleus were first identified by
phase contrast microscopy and the images thereby attained compared with
the fluorescent microscopic pictures to determine the intracellular
localization of oligonucleotides.
Immunofluorescence Detection of mAChR Subtypes in
Antisense-treated Cells--
Cultured neonatal rat ventricular
myocytes, treated with antisense oligonucleotides directed against
m1 mAChR mRNA, were probed by immunocytochemistry using
m1 and m2 mAChR subtype-specific antibodies and
the ABC Elite kit from Vector Laboratories. Briefly, cultured
ventricular myocytes were treated with 200 nM antisense oligonucleotides and 8 µg/ml Lipofectin in serum-free culture medium,
and maintained at 37 °C in a 5% CO2 incubator for
18 h. At this time, the existing medium was removed and replaced
with normal serum-supplemented culture medium. Twenty-four to 48 h later, cells were fixed with a 50% acetone:50% methanol solution for
5 min and subsequently permeabilized and blocked with PBS containing
5% goat serum, 0.3% Triton X-100, and 3% bovine serum albumin. Cells
were then exposed to affinity-purified rabbit polyclonal antibodies
specific for the m1 and m2 mAChRs (R&D Ab, used
at 1:1000 dilution) in GPBS for 24 h at 4 °C. Excess primary
antibody was removed by repeated washing with GPBS and the cells were
exposed to a biotinylated goat anti-rabbit secondary antibody, used at 1:100 dilution, for 1 h at room temperature. Following the removal of excess secondary antibody, the cells were incubated with avidin DH
and biotinylated horseradish peroxidase H, premixed according to
manufacturer's instructions. After washing, cells were stained by
incubation for 5 min with a peroxidase substrate solution containing an
equal volume of 0.02% hydrogen peroxide and 0.1% diaminobenzidine tetrahydrochloride made in 0.1 M Tris buffer, pH 7.2. Following washing and air drying, the cells were placed on coverslips
with Mowiol (Calbiochem) and viewed first using fluorescence microscopy to identify nuclear fluorescein staining, and then under the light microscope to examine peroxidase staining.
Measurements of Phosphoinositide Hydrolysis--
Inositol
phosphate (IP1) accumulation was assessed as a measure of
phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis using
modifications of previously described procedures (25). Briefly,
ventricular myocytes were cultured in 12-well tissue culture dishes for
5 days, at which time the cells were incubated with normal growth
medium containing 2 µCi/ml [3H]myoinositol (Amersham
Pharmacia Biotech) for another 36 h. The cells were rinsed twice
with a physiological saline solution (PSS) which contained
(mM): NaCl 118, KCl 4.7, CaCl2 3, MgSO4 1.2, KH2PO4 1.2, glucose 10, EDTA 0.5, and HEPES 20 (pH 7.4). The cells were then incubated with 10 mM LiCl in PSS for 30 min at 37 °C, followed by
incubation with the indicated concentrations of drugs for another 30 min at 37 °C. The reactions were terminated by removing the PSS
bathing the cells, followed by the addition of 0.6 ml of ice-cold methanol to each well. The cells were scraped from the wells and transferred to centrifuge tubes, to each of which was added 0.3 ml of
chloroform and 0.25 ml of water. The tubes were vortexed vigorously to
break up the cells, after which 0.3 ml each of chloroform and water was
added. The tubes were again vortexed and centrifuged at 1500 × g for 10 min, after which 0.7 ml of the upper phase was
withdrawn and added to 2.3 ml of water in a conical tube. Approximately
175 mg of Dowex AG1 × 8 anion exchange resin (Bio-Rad) was added
to each tube and vortexed. The gel was allowed to settle and the
supernatant poured off. The resin was then washed four times with 3 ml
of water. IP1 was extracted by the addition of 0.5 ml of
200 mM ammonium formate/100 mM formic acid to
each tube. After vortexing and allowing to settle, 0.4 ml of the
supernatant was removed and placed into a 20-ml scintillation vial.
This procedure was repeated, and 0.5 ml of supernatant was removed and
combined with the other extract. Ten ml of scintillation fluid was
added to each combined extract and counted. To account for differences in cell number between different wells, the counts obtained for each
extract were divided by the total radioactivity present in the
respective lipid fraction.
Dual Effects of mAChR Activation on the Rate of Spontaneous
Contractions--
In cardiac cells, each contraction is dependent on a
transient elevation of [Ca2+]i. Thus, we recorded
the frequency of fura-2-reported Ca2+ transients in
spontaneously beating cultured neonatal rat ventricular myocytes as a
measure of the contraction rate. Fig.
1A shows the effect of 500 nM CCh in the perfusion solution on the frequency of
spontaneous Ca2+ transients. At this concentration, CCh
immediately and completely abolished the spontaneous contractions as
evidenced by the lack of Ca2+ transients. Washout of CCh
resulted in the reappearance of Ca2+ transients,
demonstrating the reversible nature of this inhibitory response. In
most cases, the decrease in rate was accompanied by a decrease in the
diastolic Ca2+ concentration. This negative chronotropic
response to CCh was observed at all concentrations (>10
nM) tested; however, at higher CCh concentrations (>1
µM) there was often an attenuation of the response in the
continued presence of agonist. In experiments in which the CCh-induced
decrease in rate was measured, only the response in the first 2 min
after the addition of drug was taken into account for analyses. During
this time no appreciable desensitization or rebound of the response
occurred.
In a number of cardiac cell types, the stimulatory effects of mAChR
stimulation are only seen after pretreatment with PTx. To determine
whether CCh could also have stimulatory effects on the rate of
spontaneous contractions in cultured neonatal rat ventricular myocytes,
cells were treated with 100 ng/ml PTx for 18-24 h to inactivate the
Gi/Go protein and thus abolish the CCh-induced negative chronotropic response. Under these conditions, exposure of
cells to higher concentrations of CCh resulted in a
concentration-dependent, reversible increase in the rate of
spontaneous contractions (Fig. 1B). The increase in rate was
often accompanied by an increase in the diastolic Ca2+
concentration. The concentration-response curves for the CCh-induced negative and positive chronotropic responses are shown in Fig. 1C. A comparison of the curves for the two responses
demonstrates that CCh is more efficacious in eliciting the inhibitory
response (EC50 = 35 nM) as compared with the
stimulatory response (EC50 = 26 µM). The
maximal inhibitory response on the rate of spontaneous contractions was
a complete abolition of the heartbeat (100% inhibition) and was
achieved with concentrations of CCh
To determine whether the dual and opposite effects of CCh on the rate
of spontaneous contractions are both mediated through cell surface
mAChRs, the non-selective muscarinic antagonist, atropine, was used to
inhibit the observed responses. In the presence of 100 nM
atropine, the inhibitory effect of 500 nM CCh on the rate
of spontaneous contractions was completely abolished (Fig. 2A). Similarly, atropine
inhibited the CCh-induced positive chronotropic response. In cells
treated with 200 ng/ml PTx for 24 h, 50 µM CCh
increased the rate of spontaneous contractions by 34 ± 3 beats/min (n = 5). In the presence of 100 nM atropine, however, 50 µM CCh produced a
change in the rate of spontaneous contractions of
One hallmark of the dual effects of muscarinic activation on the
heartbeat obtained from previous studies on various cardiac tissue
preparations is that the two responses can be differentiated by
different muscarinic agonists. It has been reported that while CCh and
acetylcholine are able to elicit both the inhibitory and stimulatory
effects, the m2 mAChR-selective agonist, oxotremorine, is
able to elicit only the inhibitory response (4, 26). Therefore, we
determined whether oxotremorine could similarly differentiate between
the negative and positive chronotropic effects of mAChR activation in
the cultured ventricular myocytes. In three separate experiments,
addition of 1 µM oxotremorine to the superfusion buffer
completely abolished spontaneous contractions in an atropine-sensitive manner (Fig. 3A). In cells
pretreated with 100 ng/ml PTx, however, oxotremorine was unable to
mimic the CCh-induced positive chronotropic response at any
concentration tested (Fig. 3B; n = 4). These
results establish that oxotremorine differentiates between the dual
effects of mAChR stimulation on the heartbeat in a manner similar to
that found in other cardiac cell preparations.
Expression of mAChR Subtypes in Cultured Ventricular
Myocytes--
We investigated the possible expression of multiple
mAChR subtypes in the cultured heart cells using the highly sensitive and specific technique of RT-PCR. A potential problem with these studies arose from the fact that, as is typical with primary cultures, the cultured neonatal rat ventricular myocyte preparation was always
found to be contaminated by a population of non-myocyte cells most of
which are fibroblasts. This contamination was evident even after the
use of effective procedures such as preplating to limit the initial
amount of fibroblasts, and exposure to Functional Inhibition of mAChR Subtypes with Antisense
Oligonucleotides--
To determine which mAChR subtype mediates the
CCh-induced positive chronotropic response in cultured neonatal rat
ventricular myocytes, antisense oligonucleotides were used to
specifically and selectively reduce the expression of the different
mAChR subtypes. Based on the results obtained from the single-cell
RT-PCR experiments, fluorescein-tagged antisense oligonucleotides
targeted against mRNA for the m1, m2, and
m4 mAChRs were designed and tested for their ability to
inhibit the CCh-induced positive chronotropic response in cultured
ventricular myocytes. The cells were treated with 200-500
nM fluorescein-tagged oligonucleotide in the presence of
Lipofectin and functional assays carried out 48-72 h later. Measurements were only made in cells that demonstrably exhibited nuclear fluorescence at this time. In cells not treated with
oligonucleotide, 300 µM CCh resulted in an increase in
rate of 48 ± 9 beats/min (n = 3) (Fig.
6). This response was not inhibited in
cells treated with antisense oligonucleotides directed against the
m2 or m4 mAChRs, which responded to 300 µM CCh with increases in rate of 49 ± 15 beats/min
(n = 4) and 49 ± 12 beats/min (n = 3), respectively. Treatment of cells with antisense oligonucleotides
targeted against the m1 mAChR, however, resulted in a
significant reduction in the CCh-induced increase in single-cell
contraction rate. In these cells, 300 µM CCh resulted in
an increase in rate of only 16 ± 2 beats/min (n = 5) (Fig. 6). These results indicate that the CCh-induced positive
chronotropic response observed in cultured neonatal rat ventricular
myocytes is mediated through the m1 mAChR. A number of
control experiments were performed to demonstrate the specificity of
the antisense methods applied here. Immunocytochemistry was performed,
using m1- and m2-specific primary antibodies
and the ABC Elite kit from Vector Labs, on cells treated with
m1 antisense oligonucleotides to monitor changes in protein
expression. Fig. 7 (A and
C) illustrates peroxidase staining for control cells treated
with either m2 or m1 mAChR primary antibody,
respectively. In cells treated with antisense oligonucleotides to
m1 mAChR protein, a significant decrease in m1
receptor staining was observed (Fig. 7D), whereas no change
in m2 receptor staining was observed (Fig. 7B).
In functional experiments, the anti-m2 mAChR
oligonucleotides were able to inhibit the CCh-induced negative
chronotropic response in contrast to their lack of effect on the
positive chronotropic response (data not shown). Additionally, the
anti-m1 mAChR oligonucleotide was found to be ineffective
in inhibiting isoproterenol-mediated increase in contraction rate, even
though this latter response could be inhibited by an antisense
oligonucleotide directed against Gs
Previous studies have demonstrated that agonist binding to the
m1 mAChR leads to the activation of Gq protein
(27). We, therefore, tested the hypothesis that the Gq Role of the PI Hydrolysis Pathway in the CCh-induced Positive
Chronotropic Response--
Studies with cloned mAChRs have shown that
the m1 mAChR preferentially couples to PLC-mediated PI
hydrolysis when transfected into cells. Also, it has been demonstrated
that stimulation of mAChRs can induce PI turnover in cardiac cells
(28). We therefore studied the possible involvement of this signal
transduction pathway in the CCh-induced positive chronotropic response
in cultured ventricular myocytes. In 6-day-old cultures, application of
CCh resulted in a concentration-dependent increase in the
accumulation of IP (Fig. 8A).
The half-maximal concentration of CCh required for this response was 17 µM, while a maximum response of a 3-fold increase in IP
was observed with 300 µM CCh. The increase in IP accumulation induced by 150 µM CCh was inhibited by 1 µM atropine, indicating its mediation through cell
surface mAChRs (Table II). To determine
whether a cause and effect relationship exists between CCh-induced PI
hydrolysis and the positive chronotropic response, we used two putative
inhibitors of PLC-mediated PI hydrolysis, neomycin and U73122.
Pretreatment of cells with 500 µM neomycin or 5 µM U73122 resulted in a complete blockade of the positive chronotropic response induced by 300 µM CCh (Fig.
8B). The effect was recovered upon washout of neomycin or
U73122 in all cases. At these concentrations, both neomycin and U73122
were shown to inhibit the increase in IP accumulation induced by 150 µM CCh (Table II). In contrast, 500 µM
neomycin or 5 µM U73122 did not inhibit either the CCh-induced
negative chronotropic response, or the isoproterenol-induced positive
chronotropic response, in cultured ventricular myocytes (Table I).
Dual Effects of CCh on the Heartbeat--
The phenomenon in which
activation of cardiac mAChRs produces dual and opposite effects on the
heartbeat has been described in almost all cardiac cell types in
virtually every species in which this question has been addressed. For
example, muscarinic agonists have been reported to have both negative
and positive inotropic effects in guinea pig and rat atria (3), canine
and guinea pig ventricular muscle (2, 26), as well as in canine Purkinje fibers (29). In addition, negative and positive chronotropic responses to mAChR stimulation have been observed in chick sinoatrial node (30) and canine Purkinje fibers (31). Although the negative inotropic and chronotropic effects of mAChR receptor activation are the
best well known and considered physiologically relevant, the remarkable
conservation of stimulatory responses to muscarinic agonists in all
cardiac cell types and across species argues strongly for a
physiological role for this phenomenon. What this physiological role
may be is not altogether clear at present, although it has been
suggested that a stimulatory component of mAChR activation on the
heartbeat may act in an autoregulatory capacity to prevent excessive,
and thus detrimental, inhibition of the heartbeat by the vagus nerve
(4). In this regard, it is interesting to note that the phenomenon
whereby an agonist produces dual and opposite effects on the heartbeat
is not unique to the mAChR system but is in fact prevalent among other
receptor systems that are able to influence the heartbeat. For example,
both inhibitory and stimulatory effects on the heart have been reported
to occur with the stimulation of cardiac adenosine receptors (32),
P2-purinoceptors (33), and
A consistent finding relating to the stimulatory effects of mAChR
activation on the force and frequency of the heartbeat is that these
responses are insensitive to PTx and invariably require much higher
concentrations of agonist than do the inhibitory responses (4). Two
competing hypotheses have been invoked to explain these observations
(35). First, the two responses could be mediated through a single
mAChR, which couples with different efficiency to two different G
proteins. Alternatively, the two responses could be mediated through
two different mAChR subtypes. In the latter model, the inhibitory
response to cholinergic agonists is proposed to be mediated through the
m2 mAChR coupled to a Gi/Go protein
while the stimulatory response is mediated through a less abundant
mAChR subtype, other than m2, coupled to a PTx-insensitive G protein. In this work, cultured neonatal rat ventricular myocytes have been used as a cardiac cell model system to investigate these two
hypotheses. These cells were found to exhibit dual responses to mAChR
activation, responding to low concentrations of CCh (EC50 = 35 nM) with a negative chronotropic response, whereas,
after PTx treatment, they exhibited a positive chronotropic response to
higher concentrations of CCh (EC50 = 26 µM).
Further, the m2 mAChR selective agonist, oxotremorine, was
able to induce a negative but not a positive chronotropic response in
these cells. These findings indicate that these cells exhibit both
negative and positive responses to mAChR stimulation in a manner
similar to what has been observed in other cardiac cell preparations
and thus justifies their use in the present study.
Expression of mAChR Subtypes in Cultured Ventricular
Myocytes--
It has been a long held view that cardiac cells express
only the m2 mAChR on their surface membranes (27). This
view was supported by Northern blot experiments in which it was shown
that mRNA for the m2 mAChR, but not other mAChR
subtypes, could be detected in rat atrial preparations (5). This result
is difficult to reconcile with the fact that the atrial preparations
used for these studies presumably contained cell types, other than
cardiac cells, such as smooth muscle cells, fibroblasts, and nerve
cells, some of which have been found to possess mAChR subtypes other than m2 (6, 27). Thus, the detection of mRNA for only
the m2 mAChR in this preparation using Northern blot
analysis suggests a limit to the sensitivity of this technique in
detecting small amounts of mRNA. This possibility is supported by
recent experiments in which mAChR subtypes that were missed by Northern
blot studies were detected by use of RT-PCR in guinea pig ventricular
myocytes (7). In addition, work in this laboratory has demonstrated, through the use of single-cell RT-PCR, the existence of m1
and m2 mRNA in acutely isolated ventricular myocytes
from adult rat heart (10).
In this work, using the highly sensitive technique of RT-PCR, we found
that cultured neonatal rat ventricular myocytes express mRNA for
the m1, m2, and, possibly, m4
mAChRs. Furthermore, the presence of m1 and m2
mAChR protein on the surface membranes of the cultured ventricular
myocytes were confirmed by immunofluorescence. The finding of mRNA
for the m1 and m2 mAChRs in these cardiac cells
is in agreement with recent work in which both these subtypes were
found in adult guinea pig and adult rat ventricular myocytes (7, 10).
In our experiments we were also able to occasionally detect mRNA
for the m4 mAChR. The inconsistent nature of this observation could reflect a lower abundance of this mRNA when compared with those for the m2 and m1 mAChRs.
Clearly, further work is needed to establish whether the cultured
neonatal rat ventricular myocytes do indeed express the m4
mAChR. It is noteworthy, however, that in embryonic chick ventricle a
mAChR subtype termed chick m4, which has high homology to
the mammalian m4 mAChR mRNA, has been found to be
expressed (36).
Use of Antisense Oligonucleotides to Assess mAChR Subtypes Linked
to Negative and Positive Chronotropy--
The identification of
multiple mAChR subtypes in cultured neonatal rat ventricular myocytes
provides a rational basis for the hypothesis that the CCh-induced
negative and positive chronotropic responses are mediated through
different mAChR subtypes. The absolute determination of this question,
however, requires the use of agents that are able to selectively and
specifically inhibit the function of different mAChR subtypes. The use
of "selective" antagonists to mAChR subtypes in this role has
yielded conflicting results. Although data obtained using selective
mAChR subtype antagonists generally agree that the inhibitory effects
of mAChR stimulation on the heartbeat are mediated through the
m2 mAChR, the situation is not so clear for the stimulatory
response. Thus, it has been reported that "low affinity" mAChR
responses in cardiac cells are mediated through m1 mAChRs
(7, 10, 31), m2 mAChRs (3, 35), m3 mAChRs (37),
or through receptors that are neither m1 nor m2
(38).
In this study we have used antisense oligonucleotides to selectively
inhibit expression of specific mAChR subtypes in cultured rat
ventricular myocytes. These agents have found widespread use as
specific inhibitors of gene expression in a variety of in
vitro systems as well as in vivo (39). A number of
factors have been identified as being important in determining the
efficacy of antisense oligonucleotides in inhibiting gene expression.
These include the stability of the oligonucleotide, its intracellular
concentration, and its intracellular distribution. Phosphorothioate
oligonucleotides were used in this study since this modification has
been found to render the oligonucleotides nuclease-resistant, thus
enhancing their stability, without interfering with their hybridization properties (40). To assess uptake and intracellular distribution, oligonucleotides were tagged with fluorescein and observed within cells
by fluorescence microscopy. To allow for mAChRs already expressed on
the cell surface at the time of transfection to turnover, functional
assays were performed 48-72 h after transfection. At this time,
however, only 30% or less of the cells exhibited a nuclear
localization of oligonucleotide. Thus, a crucial advantage of the
fluorescence-based method described here is that it allowed for the
determination of the efficacy of antisense treatment only on the single
cells that exhibited a nuclear localization of oligonucleotide.
Fluorescein-tagged oligonucleotide antisense to mRNA for the
m1, but not the m2 or m4 mAChRs
inhibited the positive chronotropic response elicited by 300 µM CCh. Importantly, 1) cells treated with
anti-m1 mAChR oligonucleotides displayed decreased staining with m1-specific antibodies while showing no change in
m2 staining, 2) the anti-m1 mAChR
oligonucleotide had no effect on isoproterenol-induced increase in
contraction rate, and 3) the anti-m2 mAChR oligonucleotide was able to inhibit the CCh-induced negative chronotropic response. These results demonstrate the specificity of the antisense treatment and indicate that the CCh-induced negative and positive chronotropic responses are mediated through m2 and m1
mAChRs, respectively. We suggest that the stimulatory effects of mAChR
activation on the force and frequency of the heartbeat observed in
other cardiac cell preparations could be similarly mediated through
m1 mAChRs. Recent data in agreement with this conclusion
are provided by experiments conducted on adult guinea pig and rat
ventricular myocytes in which activation of pharmacologically
identified m1 mAChRs were associated with an increase in
the L-type Ca2+ current and an increase in the
magnitude of cytosolic Ca2+ transients, respectively (7,
10).
Involvement of Gqa Activation and PI Hydrolysis in the
CCh-induced Positive Chronotropic Response--
In other cell systems,
m1 mAChRs are known to couple to and activate
Gq protein (27). Involvement of Gq
It has long been suggested that the products of PI hydrolysis may be
the second messengers responsible for mediating the stimulatory effects
of muscarinic agonists on the heartbeat. This suggestion is based on a
number of factors. 1) The concentrations of muscarinic agonist required
to elicit PI turnover (EC50 = 17 µM) are high and comparable to those required to elicit positive inotropic and
chronotropic responses (EC50 = 26 µM), 2)
similar to mAChR-mediated stimulatory effects on the heartbeat, the
stimulation of PI hydrolysis by muscarinic agonists is insensitive to
PTx, and 3) other receptor systems that couple to PI hydrolysis in the
heart also generally have stimulatory effects on the rate and force of
the heartbeat. Despite this obvious correlation between mAChR
agonist-induced PI turnover and stimulatory effects on the heartbeat,
there is scant evidence establishing a cause and effect relationship
between the two events. Here, we attempted to determine whether such a relationship existed by observing the effects of blockade of PI hydrolysis on the CCh-induced positive chronotropic response. At high
concentrations, neomycin blocks PI turnover by binding to
phosphatidylinositol 4,5-bisphosphate and preventing its hydrolysis by
PLC (41). Consistent with this, 500 µM neomycin blocked
CCh-induced PI turnover by over 80% in the cultured ventricular
myocytes. More interestingly, neomycin also completely blocked the
CCh-induced positive chronotropic response in these cells, suggesting
the direct involvement of a product of the PI hydrolysis pathway in generating this response. Consistent with this notion, another putative
inhibitor of PLC-mediated PI hydrolysis, U73122 (42), blocked both the
positive chronotropic response, and PI turnover induced by 300 µM CCh. One concern relating to these results relates to
the specificity of neomycin and U73122 in inhibiting PLC-mediated PI
hydrolysis. Conceivably, these agents could inhibit other second messenger pathways that might be important in mediating the stimulatory effects of mAChR activation on the heartbeat (41, 42). However, in the
presence of 500 µM neomycin sulfate or 5 µM
U73122, these drugs had no effect on the ability of 500 nM
CCh or 1 µM isoproterenol to produce a decrease or
increase in spontaneous beating rate, respectively.
Finally, what are the possible mechanisms by which m1 mAChR
activation can elicit a positive chronotropic response? Stimulation of
m1 mAChR has been found to inhibit different types of
K+ channels through a pathway involving PLC activation and
direct tyrosine phosphorylation (43, 44). An inhibition of
K+ channels could presumably depolarize resting membrane
potential sufficiently to increase automaticity and produce a positive
chronotropic effect. Consistent with this idea, our recent preliminary
results indicate that 300 µM CCh significantly prolongs
the action potential duration and depolarizes the membrane potential in
PTx-pretreated neonatal ventricular myocytes. The signaling mechanisms
for these effects are currently under investigation.
We thank Dr. Allan Levey (Department of
Neurology, Emory University, Atlanta, GA) for the generous gift of
antibodies to muscarinic receptor subtypes.
*
This work was supported by National Institutes of Health
Grant HL-33333 and by American Heart Association/New York State
Affiliate Grant-in-aid 94-421.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom all correspondence and reprint requests should be
addressed. Tel.: 716-275-3381; Fax: 716-244-9283; E-mail: sheus{at}pharmacol.rochester.edu.
The abbreviations used are:
mAChR, muscarinic
acetylcholine receptor; PTx, pertussis toxin; RT, reverse
transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered
saline; PSS, physiological saline solution; IP, inositol phosphate; PLC, phospholipase C; PI, phosphoinositide; GPBS, PBS containing normal
goat serum.
Signaling Mechanisms Underlying Muscarinic Receptor-mediated
Increase in Contraction Rate in Cultured Heart Cells*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
protein. Finally, inhibition of CCh-induced phosphoinositide hydrolysis
with 500 µM neomycin or 5 µM U73122
completely abolished the CCh-induced positive chronotropic response.
These results are consistent with the stimulatory effects of mAChR
activation on the rate of contractions in cultured ventricular myocytes
being mediated through the m1 mAChR coupled through
Gq to phospholipase C-induced phosphoinositide hydrolysis.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-irradiation from a cesium source 24 h after the
culture. This treatment has been found to limit proliferation of
nonmyocardial cells while having little effect on the properties of
myocytes (14). Cells were washed every second day and used for
experiments after 4-14 days in culture.
and Gs protein were
designed based on their published sequences. Phosphorothioated, 20-mer oligonucleotides synthesized using phosphoramidite chemistry and purified by gel filtration were obtained from the Midland Certified Reagent Co. (Midland, TX). The oligonucleotides were further modified by the addition of a 3'-amino linker arm to allow their labeling with
fluorescein isothiocyanate (Molecular Probes). The oligonucleotides were each designed to contain comparable amounts of pyrimidines and
purines. The uniqueness of the sequences targeted by each of the
oligonucleotides used in this study was determined by comparing the
targeted sequence against sequences found in GenBank and other data
bases using BLAST (21). The antisense oligonucleotide sequences used
and the positions in the published mRNA sequences (17, 18, 22, 23)
to which they are targeted are as follows.
20 °C. For transfection of cells, 200-500 nM of fluorescein-tagged oligonucleotides and 8 µg/ml Lipofectin (Life Technologies, Inc.) were each diluted in 100 µl of serum-free Dulbecco's modified Eagle's medium prewarmed to
37 °C. The two solutions were then combined and incubated at room
temperature for 20 min. For each transfection, 0.8 ml of serum-free
medium was added to each tube containing the Lipofectin-oligonucleotide complexes and the resulting mixture overlaid onto the cultured cells.
Unless otherwise stated, the cells were incubated with the
Lipofectin-DNA complexes for 18 h at 37 °C in a CO2
incubator, after which time this solution was removed and replaced with
serum-containing normal growth medium.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (26K):
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Fig. 1.
Dual effects of CCh on contraction rate in
single cultured cardiomyocytes. A, effect of 500 nM CCh on the frequency of spontaneous Ca2+
transients in a single beating cultured ventricular myocyte.
B, effect of 300 µM CCh on spontaneous
contractions in a cell from a chamber treated with PTx (100 ng/ml for
18-24 h). Solid bars above the tracings
represent the period of application of drugs. C,
concentration-response curves for the CCh-induced decrease
(open circles) and increase (open
squares) in spontaneous contraction rate. Data points and
error bars represent means ± S.E. from at least three separate
experiments.
500 nM. For the positive chronotropic response, the threshold concentration for the
observation of a response was 5 µM CCh and the maximum
response was obtained with 300 µM CCh. At this
concentration, CCh increased the rate of spontaneous contractions from
a control value of 59 ± 6 beats/min (n = 12) to a
value of 119 ± 8 beats/min (n = 12). After two
minutes of wash, the rate of spontaneous contractions returned toward
control rates with a value of 73 ± 7 beats/min, thus
demonstrating the reversible nature of the positive chronotropic response.
3 ± 3 beats/min, indicating a complete block of the positive chronotropic response (Fig. 2B). Thus, both the CCh-induced negative and
positive chronotropic effects are mediated through cell surface
mAChRs.
View larger version (38K):
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Fig. 2.
Effects of atropine on the dual chronotropic
response to CCh. A, representative tracing showing the
abolition of the decrease in contraction rate induced by 500 nM CCh after preincubation of cells with 100 nM
atropine. Solid bars represent the period of
application of the different drugs. Similar results were obtained in
three other experiments. B, bar chart showing the effect of
100 nM atropine on the increase in contraction rate induced
by 50 µM CCh in PTx-treated cells. Values represent
means ± S.E. with the number of experiments shown in
parentheses.
View larger version (41K):
[in a new window]
Fig. 3.
Effects of oxotremorine on single-cell
contraction rate. A, representative tracing showing the
inhibition of spontaneous Ca2+ transients induced by 1 µM oxotremorine and the blockade of this response by 100 nM atropine. Similar results were obtained in two other
experiments. B, effect of 300 µM oxotremorine
on the rate of spontaneous contractions in a cell from a chamber
treated with 100 ng/ml PTx for 24 h. Solid
bars above the tracings represent periods of application of
drugs. Similar results were obtained in three other experiments.
-irradiation to inhibit their
proliferation thereafter (14). These methods resulted in cultures which
identifiably contained about 80-90% ventricular myocytes after 2 weeks in culture. Nevertheless, due to the extreme sensitivity of the
RT-PCR procedure, it was recognized that even this low level of
contaminating cells could potentially be the source of a confounding
artifact by contributing to the mAChR subtypes identified. For this
reason, it was necessary to use a method capable of identifying mAChR
subtypes that are expressed in the ventricular myocytes to the
exclusion of those expressed in the contaminating fibroblasts. This was
achieved in two different ways. First, single-cell RT-PCR was carried
out on mRNA obtained by sucking up the contents of single
ventricular myocytes into glass micropipettes. Using this technique, we
consistently obtained amplification products corresponding to the
m1 and m2 mAChRs (Fig. 4A). In two experiments, we
obtained an amplification product corresponding to the m4
mAChR, however, this observation could not be reproduced consistently.
We did not observe products corresponding to the m3 or
m5 mAChRs in any experiment. The identity of the m1 band was confirmed by Southern blot (Fig. 4B)
and by sequencing the PCR products (data not shown). Controls for the
single-cell RT-PCR reactions were provided by experiments in which
reverse transcriptase was omitted during the reverse transcription step (Fig. 4A). In a second approach, immunofluorescence was used
to confirm the results obtained from the single-cell RT-PCR
experiments. Fig. 5 shows confocal images
obtained from immunofluorescence studies when cells were incubated with
either no primary antibody (A), or primary antibodies
against the m1 and m2 mAChRs (B and C, respectively). Staining of the cell membrane was seen for
both m1 and m2 mAChR antibodies. Thus, the
results obtained by immunocytochemistry are consistent with the
single-cell RT-PCR data, and together these provide strong evidence for
the existence of multiple mAChR subtypes on the surface membranes of
cultured neonatal rat ventricular myocytes.
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Fig. 4.
Expression of mAChR subtypes in cultured
ventricular myocytes. A, amplification products
obtained by RT-PCR carried out on mRNA derived from single cultured
neonatal rat ventricular myocytes, using mAChR subtype-specific PCR
primers. B, Southern blot of m1 and
m2 RT-PCR products from single cultured neonatal rat
ventricular myocytes employing a DNA probe selective for m1
cDNA.
View larger version (6K):
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Fig. 5.
Detection of mAChR protein on cultured
ventricular myocytes by immunofluorescence. Confocal microscopy
images obtained from immunofluorescence experiments where cells were
incubated with no primary antibody (A), anti-m1
mAChR antibody (B), or anti-m2 mAChR antibody
(C).
protein mRNA
(data not shown).
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Fig. 6.
Effects of mAChR antisense oligomers on
CCh-induced increase in contraction rate. Bar graph showing the
effect of fluorescein-tagged antisense oligonucleotides directed
against mRNA for the m1, m2, and
m4 mAChRs, and Gq protein on the increase in
contraction rate induced by 300 µM CCh in PTx-treated
cultured neonatal rat ventricular myocytes. Values represent means ± S.E. with the number of experiments shown in parentheses
(*, p < 0.05 when compared with control using
Student's unpaired t test).
View larger version (34K):
[in a new window]
Fig. 7.
Effect of m1 antisense
oligonucleotides on m1 and m2 receptor
levels. Representative figures of immunocytochemistry performed on
cultured neonatal rat ventricular myocytes treated with m1
antisense oligonucleotides: A, control cell treated with
m2-specific antibody; B, m1
antisense-treated cell stained with m2-specific antibody;
C, control cell treated with m1-specific
antibody; D, m1 antisense-treated cell stained
with m1-specific antibody.
protein was responsible for transducing the mAChR-mediated increase in
single-cell contraction rate in response to CCh using an antisense
approach. Antisense oligonucleotides directed against the
Gq protein were also effective in significantly
inhibiting the CCh-induced positive chronotropic response. In cells
treated with anti-Gq oligonucleotides, 300 µM CCh induced an increase in the rate of spontaneous
contractions of 18 ± 5 bpm (n = 7). This result
suggests the involvement of Gq protein in mediating the
CCh-induced positive chronotropic response (Fig. 6). To demonstrate
that inhibition of the Gq-mediated pathway by antisense
oligonucleotides does not interfere with isoproterenol-mediated
activation of Gs or carbachol-mediated activation of
Gi protein pathways, spontaneously beating cells treated
with Gq protein-specific oligonucleotides were perfused with either 1 µM isoproterenol or 500 nM carbachol. Table
I demonstrates that blockade of the
Gq-mediated pathway has no effect on either the ability
of isoproterenol to produce an increase in spontaneous beat rate via
Gs activation, or the ability of carbachol to decrease the spontaneous beat rate via Gi activation.
Effects of inhibitors of PI hydrolysis and Gq antisense
oligonucleotides on carbachol-mediated inhibition of beat rate and
isoproterenol mediated stimulation of beat rate
antisense oligonucleotides in the
presence of 8 µg/ml lipofectin, and the effects of 500 nM
carbachol (CCh) and 1 µM isoproterenol (Iso) on beat rate
were monitored. 1 µM atropine = Atr; 1 mM propranolol = Prop. Values represent means ± S.E. with n representing the number of experiments performed. *,
p < 0.05 when compared to control using Student's
unpaired t test.
View larger version (20K):
[in a new window]
Fig. 8.
Involvement of PI hydrolysis in CCh-induced
increase in contraction rate. A, concentration-response
curve for CCh-induced phosphoinositide hydrolysis in cultured neonatal
rat ventricular myocytes. Data points represent means ± S.E.
(n = 3 for each point). B, bar graph showing
the effect of pretreatment of cells with 500 µM neomycin
or 5 µM U73122 on the increase in spontaneous contraction
rate induced by 300 µM CCh. Values are means ± S.E.
with the number of experiments shown in parentheses.
Effect of various pharmacological agents on total PI production
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-adrenergic receptors (34).
Given this apparent ubiquity, it is reasonable to speculate that the
phenomenon of agonists having dual effects on the heartbeat is a common
mechanism employed by cardiac cells to regulate or fine-tune the
effects of various agonists on the chronotropic and inotropic state of
the heart.
protein
in the CCh-induced increase in spontaneous beat rate in cultured
neonatal rat ventricular myocytes was investigated through the use of
Gq-specific antisense oligonucleotides. Inhibition of
Gq protein expression resulted in a significant decrease
in the ability of CCh to stimulate spontaneous beat rate, suggesting
the involvement of this protein in the stimulatory pathway. In
contrast, blockade of the Gq-mediated pathway in
cultured ventricular myocytes had no effect on the ability of those
cells to activate Gs protein in response to isoproterenol treatment or to activate Gi protein in
response to low concentrations of CCh.
ACKNOWLEDGEMENT
FOOTNOTES
Advanced predoctoral fellow of the Pharmaceutical Research and
Manufacturers of America Foundation during the tenure of this work.
REFERENCES
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Abstract
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Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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