From the * Department of Pharmacology and Institute for Cardiovascular Sciences, Georgetown University, Washington, DC 20007; and Departments of Physiology and Medicine; University of California Los Angeles, School of Medicine; Los Angeles, California 90095
We have produced transgenic mice which overexpress cardiac Na+-Ca2+ exchange activity. Overexpression has been assessed by Western blot, Northern blot, and immunofluorescence. Functional overexpression was analyzed using membrane vesicles and isolated ventricular myocytes. In whole cell clamped myocytes dialyzed
with 0.1-0.2 mM Fura-2, the magnitude of ICa and Ca2+i -transient triggered by ICa or caffeine were not significantly different in transgenic vs. control myocytes. In transgenic myocytes, activation of ICa, however, was followed by a
large slowly inactivating transient inward current representing INa-Ca. This current depended on Ca2+ release as it
was abolished when sarcoplasmic reticulum (SR) Ca2+ was depleted using thapsigargin. Cai-transients triggered by rapid application of 5 mM caffeine, even though equivalent in control and transgenic myocytes, activated larger
INa-Ca (~5 pA/pF at 90 mV) in transgenic vs. control myocytes (1.5 pA/pF). The decay rate of caffeine-induced
Ca2+i -transient and INa-Ca was 2.5 times faster in transgenic than in control myocytes. 5 mM Ni2+ was equally effective in blocking INa-Ca in control or transgenic myocytes. In 9 out of 26 transgenic myocytes, but none of the controls, Ca2+ influx via the exchanger measured at +80 mV caused a slow rise in [Ca2+]i triggering rapid release of
Ca2+ from the SR. SR Ca2+ release triggered by the exchanger at such potentials was accompanied by activation of
transient current in the inward direction. In 2 mM Fura-2-dialyzed transgenic myocytes caffeine-triggered Cai-transients failed to activate INa-Ca, even though the kinetics of inactivation of ICa slowed significantly in caffeine-treated myocytes. In 0.1 mM Fura-2-dialyzed transgenic myocytes 100 µM Cd2+ effectively blocked ICa and suppressed Cai-transients at
10 or +50 mV. Our data suggests that in myocytes overexpressing the exchanger, the
content of intracellular Ca2+ pools and the signaling of its release by the Ca2+ channel vis-à-vis the Na+-Ca2+ exchanger were not significantly altered despite an up to ninefold increase in the exchanger activity. We conclude
that the exchanger remains functionally excluded from the Ca2+ microdomains surrounding the DHP/ryanodine
receptor complex.
Regulation of Ca2+ fluxes in cardiac myocytes is a complex process involving multiple transporters, channels,
and compartments. A key transport process is that mediated by the Na+-Ca2+ exchange protein. The Na+-Ca2+
exchanger catalyzes the countertransport of three Na+
ions for one Ca2+ ion across the sarcolemmal membrane and is the major Ca2+ efflux mechanism of myocardial cells. Ca2+ extrusion by the exchanger helps bring
about muscle relaxation after contraction. Thus, while
the Ca2+ channels of sarcolemma (DHP receptors) provide the primary route for entry of Ca2+, the Na+-Ca2+
exchanger serves as a primary route for Ca2+ extrusion.
To maintain and regulate cytosolic Ca2+ concentrations
during the contraction/relaxation cycle, the activity of
the two pathways ultimately must be balanced. Although the role of the Na+-Ca2+ exchanger in the efflux of Ca2+ has been quantified and is universally accepted, the physiological role of the exchanger in the
Ca2+ influx mode has been somewhat controversial and
not generally agreed upon (Callewaert, 1992; Levesque
et al., 1994
; Lipp and Niggli, 1994
; Sham et al., 1992
).
This controversy may be related, in part, to different
levels of expression of the exchanger in different species (Sham et al., 1995b
). Ca2+ cross signaling experiments between the DHP and ryanodine receptors in
myocytes dialyzed with high concentrations of Ca2+
buffers, however, suggest that the exchanger protein
might be excluded from the "functional" microdomain
of Ca2+ surrounding the DHP/ryanodine receptor complex (Sham et al., 1995a
; Adachi-Akahane et al., 1996
),
even though immunofluorescence techniques have suggested that the exchanger is located in the t-tubular system near diadic or triadic junctures (Frank et al., 1992
;
Kieval et al., 1992
).
The availability of the cDNA clone for the Na+-Ca2+
exchanger (Nicoll et al., 1990) allows new approaches
to physiological problems regarding the exchanger.
For example, antisense oligonucleotides have been used
to "knock out" exchange activity in both cardiac and arterial myocytes (Lipp et al., 1995
; Sidzinski et al., 1995
). Transgenic mouse technology offers another opportunity to manipulate Na+-Ca2+ exchange activity. Here we
describe the production and characterization of transgenic mice overexpressing Na+-Ca2+ exchange activity
specifically in cardiac muscle. Myocytes from the transgenic mice have increased INa-Ca and were used to further probe the ability of INa-Ca and the Ca2+ channel to
trigger Ca2+ release. Even with the increased density of
INa-Ca, the exchanger failed to trigger Ca2+ release from
the sarcoplasmic reticulum (SR)1 in the physiological
voltage range.
Production of Transgenic Mice
The transgene construct (Fig. 1) consisted of the open reading
frame of the canine cardiac Na+-Ca2+ exchanger (Nicoll et al.,
1990) under the control of the
-myosin heavy chain (
-MHC)
promoter. The
-MHC promoter consisted of 4.5 kb of 5
upstream sequence plus 1 kb of the
-MHC gene encompassing exons 1 through 3 of the 5
untranslated region (Gulick et al., 1991
;
Subramaniam et al., 1991
). The presence of upstream introns and exons may improve expression of a transgene (Palmiter et
al., 1991
). Downstream from the
-MHC promoter was the SV40
transcriptional terminator to provide a polyadenylation signal.
The exchanger open reading frame was removed from pTB11
(Nicoll et al., 1990
) by digestion with EcoRV and SnaBl and ligated into the SalI site between the
-MHC promoter and the
SV40 transcriptional terminator. The SalI site had first been digested, blunted, and dephosphorylated. Proper orientation of
the exchanger insert was confirmed by restriction mapping. The
transgene was purified by GeneClean (Bio 101, La Jolla, CA) and
microinjected into the nuclei of C57Bl/6xC3HF1 mice by the
UCLA Transgenic Core Facility for transgenic mouse production.
Eight lines (A through H) of transgenic mice were generated.
Southern blot analysis was used to confirm the presence of the transgene in the mouse genome. Genomic DNA was extracted
from tail clippings, digested with EcoRI, and size fractionated on a 0.8% agarose gel. After transfer to nitrocellulose, the DNA was
probed with the 0.4-kb EcoRI fragment from pTB11. This portion of the open reading frame of the Na+-Ca2+ exchanger is derived from a region of the NCX1 gene encompassing several exons (Kofuji et al., 1994). Thus, on the Southern blot, the 0.4-kb
probe would hybridize with the 0.4-kb fragment derived from the
EcoRI-digested transgene which contains no exchanger introns.
Digestion of the endogenous mouse exchanger gene, however, with EcoRI did not produce a similar sized fragment. Prehybridization and hybridization were carried out as described previously (Li et al., 1994
).
Northern Blot Analysis
Total RNA was isolated from mouse tissues by the method of
Chomczynski and Sacchi (1987). The RNA was separated on a
denaturing 1% agarose gel and transferred onto a Hybond-N nylon filter. The same 0.4-kb cDNA fragment used in the Southern
blot analysis above was also used for Northern blot analysis. Hybridization conditions were also the same.
Western Blot Analysis
Proteins were first separated on a 7.5% gel by SDS-PAGE and
transferred onto nitrocellulose for 30 min at 100 V. Washings and antibody incubations were carried out in the presence of 1% milk. The primary polyclonal antibody () was raised against the canine cardiac exchanger and has been described previously (Philipson et al., 1988
). The secondary antibody was goat anti-rabbit
IgG coupled to horseradish peroxidase. Diaminobenzidine was
used as substrate for color development.
Indirect Immunofluorescent Labeling
Isolated mouse myocytes, either from control hearts or transgenic hearts, were fixed with 2% buffered formaldehyde for 15 min. The fixed cells were quenched in Na+ borohydrate, treated
with Triton X-100, and exposed to blocking solution and the
monoclonal antibody R3F1 (1/500 dilution) against the Na+-Ca2+
exchanger, as previously described (Frank et al., 1992; Chen et
al., 1995
). The cells were incubated with fluorescein-labeled goat
anti-mouse secondary antibody for 45 min, rinsed, and mounted on glass slides with 90% glycerol plus a photobleaching inhibitor. The confocal fluorescence microscopy was carried out with a Nikon photomicroscope equipped with a molecular dynamic confocal imaging system.
Transport Measurements in Isolated Vesicles
A crude membrane fraction was prepared for measurement of Na+-Ca2+ exchange fluxes. Mouse hearts (~100 mg) were homogenized in 1.4 ml of 560 mM NaCl, 10 mM Mops/Tris, pH 7.4, and spun in an Eppendorf centrifuge for 4 min at 11,000 rpm. The pellet was resuspended in 0.9 ml of 140 mM NaCl, 10 mM Mops/Tris, pH 7.4 and spun for 4 min at 11,000 rpm. The pellet was resuspended in 0.8 ml of the same solution and spun briefly (5 s) at 4,000 rpm to remove particulate material. The supernatant, containing Na+-loaded membrane vesicles was used directly for Ca2+ uptake measurements. The protein yield in the final fraction was identical for the control and transgenic mice.
To measure Na+ gradient-dependent 45Ca2+ uptake, 10 µl of
the supernatant was rapidly diluted into a Ca2+ uptake medium
containing 140 mM KCl, 10 µM 45Ca2+, 1 µM valinomycin, 10 mM Mops/Tris, pH 7.4. The reaction was quenched after 3 s and
then filtered. A blank was subtracted in which the uptake medium contained NaCl instead of KCl. We have used this technique extensively in the past to quantitate vesicular Na+-Ca2+ exchange (Li et al., 1991).
Isolation of Adult Mouse Ventricular Myocytes
Adult mouse ventricular myocytes were isolated according to a
previously described method (Mitra and Morad, 1985) with minor modification. After injection of heparin sodium (1,000 U/
kg, i.p.), mice were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.), hearts were quickly excised and perfused
in a Langendorff apparatus (1.2-1.6 ml/min) first with nominally Ca2+-free Tyrode's solution composed of (in mM) NaCl,
137; KCl, 5.4; HEPES, 10; MgCl2, 1; glucose, 10; pH 7.3 at 37°C
for 7 min, then with Ca2+-free Tyrode's solution containing collagenase (0.5-0.6 Us/ml) and protease (0.55 Us/ml) for 15 min, and
finally with low Na+ Tyrode's solution. The ventricular part of the
digested heart was then cut into several sections and gently agitated to dissociate cells. The freshly dissociated cells were stored
at room temperature in low Na+ Tyrode's (in mM: 52.5 NaCl, 4.8 KCl, 1.19 KH2PO4, 1.2 Mg SO4, 11.1 glucose, 10 HEPES, 145 sucrose, pH 7.4) containing 0.2 mM Ca2+ and were used for up to
10 h after isolation. In all of the electrophysiological experiments,
the transgenic mouse line H was used.
Current Recording
Ca2+ current was measured in the whole cell configuration of
the patch-clamp technique (Hamill et al., 1981) using a DAGAN
8900 amplifier (Dagan Corp., Minneapolis, MN). The patch electrodes, made of borosilicate glass capillaries, were fire-polished to have a resistance of 1.2 to 2.0 M when filled with the internal solution composed (in mM): CsCl, 110; tetraethylammonium
chloride (TEA-Cl), 30; NaCl, 10; HEPES, 10; MgATP, 5; cAMP,
0.2; K5Fura-2, 0.1-2.0 and titrated to pH 7.4 with CsOH. Inward
rectifier K+-current was suppressed by either addition of Ba2+
(0.1 mM) to or omission of K+ from the external solutions. Na+-current was mostly suppressed by addition of 10 µM tetrodotoxin to the external solution, and by including a high concentration (200 µM) of cAMP in the internal solution (Schubert et al., 1989
). Myocytes were dialyzed with 200 µM cAMP not only to enhance
ICa but also to fully activate Ca-ATPase activity through phosphorylation of phospholamban.
Generation of voltage-clamp protocols and acquisition of data were carried out using pCLAMP software (version 5.5-1; Axon Instruments, Inc., Foster City, CA). The leak currents were not digitally subtracted by the P/N method (N = 5-6) as to avoid suppression of maintained components of INa-Ca. Thus, we chose cells which had little or no leak currents. The series resistance was 1.5 to 3 times the pipette resistance and was electronically compensated through the amplifier. Sampling frequency was 0.5-2.0 kHz, and current signals were filtered at 10 kHz before digitization and storage.
Intracellular Calcium Measurements
Intracellular calcium activity was measured according to the
method described earlier (Cleemann and Morad, 1991). Ventricular myocytes were dialyzed with 0.1-2.0 mM Fura-2 via the patch
pipettes. Ultraviolet light originated from a 100 W mercury arc
lamp, was split into two beams using a mirror vibrating at 1,200 Hz, and passed through the interference filters (410 and 335 nm,
20 nm bandwidth). The fluorescent light passed through a wide-band interference filter (510 nm, 70 nm bandwidth) and was detected with a photo-multiplier. The signal from the photo-multiplier was demultiplexed (Cleemann and Morad, 1991
), yielding
two signals corresponding to the two wavelengths of excitation.
These signals were acquired simultaneously with the whole-cell
currents using pCLAMP software. Cai -transients were quantified
using FURA 2N program (Adachi-Akahane et al., 1996
).
The data collected with dual wavelength excitation of Fura-2
were analyzed to determine the intracellular Ca2+ activity ([Ca2+]i)
by the ratiometric method with a Kd value of Fura-2 for Ca2+ as
220 nM (Grynkiewicz et al., 1985). The background fluorescences (F410,bg and F335,bg) were measured after making a giga-seal
just before rupture of the membrane. Calibration measurements
were performed with samples of 50 µM Fura-2 either saturated
with 5 mM Ca2+ (F410,Ca and F335,Ca) or in free form with 10 mM
EGTA (F410,EGTA and F335,EGTA).
Drugs were dissolved in the external Tyrode's solution, and applied rapidly using a concentration-clamp device (Cleemann and Morad, 1991).
All the experiments were performed at room temperature (22-25°C).
Collagenase (type A) was purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN), Protease (type XIV, pronase E) and MgATP from Sigma Chemical Co. (St. Louis, MO),
thapsigargin and tetrodotoxin from Calbiochem Corp. (La Jolla,
CA), and K 5Fura-2 salt from Molecular Probes, Inc. (Eugene,
OR). Thapsigargin was dissolved in DMSO and stored as 103 M
stock solution. The highest (0.1%) concentration of DMSO used had no effect by itself on ICa or Cai-transients.
Transgenic Mice, Their Molecular and Ultrastructured Characterization
Transgenic mouse lines were produced to overexpress
the canine cardiac Na+-Ca2+ exchanger in mouse myocardium. The transgene used in these experiments (Fig.
1) contained the open reading frame of the canine sarcolemmal Na+-Ca2+ exchanger under the control of
the -MHC promoter. This promoter has been used
previously in transgenic experiments for cardiac-specific expression (Milano et al., 1994
; Soonpaa et al., 1994
;
Koch et al., 1995
). Eight transgenic mouse lines, called
lines A through H, were generated. The transgenic
mice were all heterozygous so that in all cases nontransgenic littermates could be used for controls. The mice
had no readily observable altered phenotype; body and
heart weights were all normal. No differences between the transgenic mouse lines were detected unless specifically mentioned below. Table I compares some electrophysiological properties of the myocytes not directly related to the exchanger activity in control and transgenic mice. The size of the myocytes (cell capacitance),
the resting Ca2+ concentrations (basal [Ca]i), the magnitude of triggered Cai-transients (
Cai), and the Ca2+
current density (ICa) were not significantly altered in
transgenic mice.
Table I.
Ca2+ Current (ICa) and Ca2+i -transient ( |
Molecular Evidence for Overexpression of the Na+-Ca2+ Exchanger
Northern blot analysis.We first analyzed RNA from control and transgenic mouse hearts for levels of exchanger transcript derived from the transgene in the
different transgenic mouse lines. The native mouse exchanger has a transcript size of 7 kb; most of the size of
the transcript is due to extensive 5- and 3
-untranslated regions with the open reading frame of the exchanger being only 3 kb. Most of the untranslated regions have been removed in the construction of the
transgene and the expected size of the transcript in this
case is 3.2 kb. Thus, it is straightforward to distinguish
the two transcripts by Northern blot analysis.
Fig. 2 A shows a Northern blot with RNAs isolated
from the hearts of transgenic mice and their nontransgenic littermates from six different lines probed with
an exchanger cDNA probe. A strong signal from RNA
isolated from the transgenic hearts is seen at 3 kb after
an exposure of only 1 h. With such short exposure
times, no signal is observed with the RNA from the control mice. A signal from the native exchanger becomes
visible at 7 kb in all lanes after longer exposures (not
shown, but see Fig. 2 B). Similar results were obtained
for transgenic mouse lines B and E. Clearly, substantial
amounts of RNA are being transcribed from the transgene.
The -MHC promoter is supposed to permit gene expression only in cardiac tissue, therefore we next assessed the tissue specificity of exchanger transgene expression. Fig. 2 B shows the results of a Northern blot
analysis using RNA isolated from the heart, lung, brain,
or skeletal muscle of transgenic mice or nontransgenic littermates from line H. In this case, a longer exposure
time was used to permit visualization of both the native
and transgenic Na+-Ca2+ exchangers. In the tissues from
the nontransgenic mice, a weak signal at 7 kb from the
native exchanger is seen only in the RNA from the cardiac tissue. (Upon longer exposure, 7-kb bands also become visible in the RNA from brain and lung.) In the
RNAs from the transgenic mice, a strong signal is seen
only with the cardiac RNA at 3 kb (transgenic exchanger) and a weak signal is seen at 7 kb (native exchanger). Not visible in the photograph, but discernible by eye, is a low level of expression of the 3-kb transgenic exchanger in the lung of transgenic mice. This was
also seen in lung RNA from other transgenic mouse
lines. Thus, the
-MHC promoter was not completely
silent in lung tissue. In one transgenic mouse line (line
E), expression of transgene transcripts were much higher in lung than in the other transgenic lines though still
many fold lower than the expression level in heart.
Line E was also the only line in which transgene transcripts could also be weakly detected in brain RNA.
Subramaniam et al. (1991)
have previously noted a low
level of
-MHC gene expression in lung tissue, specifically in the thick wall of the pulmonary veins of the
lung. Nevertheless, of those tissues tested, high levels of
transgenic Na+-Ca2+ exchanger transcript were found
only in the myocardium.
Immunoblots were performed to
assess the level of Na+-Ca2+ exchanger protein in the
hearts of the transgenic mice. The proteins of myocardial homogenate were separated by SDS-PAGE and
probed with a polyclonal antibody to the canine sarcolemmal Na+-Ca2+ exchanger. Strong immunoreactivity is seen in the transgenic hearts but not in the hearts
from control littermates (Fig. 3). The protein bands
which immunoreact have a similar pattern to that seen
with isolated canine sarcolemmal membranes, a positive control (Philipson et al., 1988). The transgenic exchanger protein bands, however, appear to be of slightly
smaller molecular weight than those of the isolated sarcolemma, perhaps due to a small difference in amount
of glycosylation. We have previously demonstrated that
glycosylation does not affect exchanger function (Hryshko et al., 1993
).
The native mouse exchanger of the control mouse hearts produces only a weak immunoreactivity under these conditions, though at the same apparent molecular weight as the transgenic exchanger protein. The strength of the immunoreactivity, however, cannot be used to estimate quantitatively the amount of exchanger overexpression. The antibody was produced using the canine heart exchanger as antigen and thus may react more weakly with the native mouse heart exchanger than with the canine exchanger encoded by the transgene. Qualitatively, however, the Western blot clearly demonstrates that a substantial amount of Na+-Ca2+ exchanger protein is being translated from transgene transcripts.
Immunofluorescence
Overexpression of the Na+-Ca2+ exchanger is also clearly
demonstrated by immunofluorescence (Fig. 4). The
confocal micrographs of the control (Fig. 4 A) and
transgenic (Fig. 4 B) myocytes were taken under identical conditions. In the transgenic myocytes, there is intense labeling of both the surface and t-tubular sarcolemma as well as the area surrounding the nucleus
which is presumably the Golgi apparatus involved in
protein trafficking. As described above, the antibody
reactions cannot be used for quantitative comparison
of exchanger expression.
We had previously found a preferential localization
of the Na+-Ca2+ exchanger in that part of the sarcolemma which forms the t-tubules in guinea pig and rat
myocytes (Frank et al., 1992). This preferential localization is much less obvious in mouse myocytes in which
strong staining of the peripheral sarcolemma is also observed. This species difference may be related to the
large amount of peripheral sarcoplasmic reticulum found
in mouse myocytes ( J.S. Frank, unpublished observations). Thus, even the Na+-Ca2+ exchangers found in
surface sarcolemma in mouse myocytes may be in close
proximity to underlying sarcoplasmic reticulum.
Two types of experimental protocols using freshly isolated ventricular myocytes and one set of experiments using a crude membrane preparation were employed to examine the exchanger activity in control and transgenic mice.
In the first set of experiments we prepared a crude
myocardial membrane fraction and assayed for Na+
gradient-dependent 45Ca2+ uptake. In a second set of
experiments, Fura-2-dialyzed myocytes were clamped at
holding potentials of 90 mV and were subjected to
rapid (<50 ms) application of 5 mM caffeine to induce
Ca2+ release from the SR to activate a transient Ni2+-sensitive inward INa-Ca (Ca2+-extrusion mode of the exchanger; Callewaert, Cleemann and Morad, 1989). In a
third set, long (1-2 s) depolarizing pulses to positive potentials were used to measure the Ca2+ influx mode
of the exchanger. The maintained outward Ni2+-sensitive current and the accompanying rise in [Ca2+]i were
quantified to represent Ca2+ transported by the exchanger (Kimura et al., 1986
; Näbauer and Morad, 1992
).
We prepared crude membrane fractions and assayed
Na+ gradient-dependent 45Ca2+ uptake. Results obtained
using hearts from transgenic mouse line H are shown
in Fig. 5. Na+-Ca2+ exchange activity is 148% higher in
vesicles from transgenic hearts than in vesicles from the
hearts of control littermates. The level of overexpression in vesicles from transgenic hearts from other mouse
lines averaged about 100%, though due to scatter, it
was not clear if there were significant differences. The
level of overexpression as assessed electrophysiologically
from line H mice (see Table II) appears to be somewhat
larger than that measured in vitro by isotope flux.
Table II.
Caffeine-induced Na-Ca Exchange Current and Cai-transient at Holding Potential of |
Fig. 6 compares the Ca2+ transients triggered by rapid (<50 ms) application of caffeine in two
isolated whole-cell clamped ventricular myocytes dialyzed with 0.2 mM Fura-2. In control mice, 5 mM caffeine triggered a Cai-transient and a slowly activating
exchanger current of about 250 pA decaying with a
time constant of 300 ms. In transgenic myocytes, although the caffeine-triggered Cai-transient was similar in magnitude, the accompanying exchanger current
was three times larger than in control mice. In addition, the kinetics of decay of both the exchanger current and Cai-transients in transgenic myocytes were two
to threefold faster than those of control myocytes. In
control as well as transgenic myocytes, 5 mM Ni2+ effectively blocked the caffeine-induced INa-Ca but allowed
the Cai-transient to develop. Fig. 6 (transgenic + Ni2+)
shows that the strong suppression of INa-Ca by Ni2+ in
transgenic mice is accompanied by a 20-fold reduction
in the rate of decay of Ca2+ transients, confirming the
prominent role of the exchanger in the Ca2+ efflux process. Comparison of the rates of decay of Cai-transients and INa-Ca suggests a two to threefold increase in the kinetics of Ca2+ extrusion in transgenic myocytes (Table
II). In 14 control and 16 transgenic myocytes dialyzed
with 100-200 µM Fura-2, the Ni2+-sensitive current (INa-Ca)
averaged about 1.6 and 5.0 pA/pF, respectively (see Table
II). Though the magnitude of Ca2+ release was not significantly different in control and transgenic myocytes, the
exchanger activity was enhanced in transgenic myocytes by all three criteria tested: the magnitude of INa-Ca, the kinetics of INa-Ca, and the rate of decay of Cai-transients.
Table II quantifies some of the parameters of INa-Ca
and the accompanying Cai-transients in transgenic and
control myocytes. The size of the caffeine-triggered Ca2+
pool was found to be equivalent in control and transgenic myocytes. Comparison of the caffeine-releasable
pool in normal and transgenic mice in the presence of
5 mM Ni2+ revealed a somewhat larger caffeine-sensitive pool in transgenic. In addition, the rate of release
of Ca2+ (an indirect indication of flux of Ca2+ through
the ryanodine receptor), though equivalent in control and transgenic mice, was enhanced in the presence of
Ni2+ only in transgenic myocytes (Table II). These findings suggest that the higher exchanger activity reduces
the cytosolic Ca2+ concentrations very rapidly in transgenic mice. It is premature to suggest possible compensatory Ca2+ adaptive pathways, as we do not have direct
data on the activity or regulation of SR and SL Ca2+
pumps, the ryanodine receptors, and, in particular,
Ca2+ storing proteins such as calsequestrin in myocytes
overexpressing the exchanger. Some indication as to
the activity of SR Ca2+ pump in transgenic myocytes was
obtained by comparing the rate of relaxation of Cai-transient, triggered by brief (50 ms) pulses of caffeine,
in Na+-free solutions. Yao and Barry (personal communication) found no significant differences in the rate of
relaxation of Ca2+-transients between control and
transgenic myocytes. Although such studies are complicated by the rate of dissociation of Ca2+ from the dye,
they nevertheless do not support compensatory enhancement of Ca2+ ATPase activity in transgenic myocytes.
Compensatory responses in transgenic mice showing
no phenotypic changes, as was the case here, are likely
to be subtle and multifaceted. Upregulation of calsequestrin, for example, could serve as a possible mechanism by which the SR could maintain its Ca2+ load in
the face of overexpression of exchanger protein. Such an idea is supported by a recent study that reports
upregulation of Ca2+ release pools in transgenic mice
overexpressing cardiac calsequestrin (Suzuki et al., 1997).
Fig. 7 illustrates ICa-gated Ca2+ release in two myocytes obtained from transgenic and non-transgenic littermates. In both cell types the magnitude of ICa and
Cai-transients were similar (see also Table I). In transgenic cells, however, ICa measured between 20 and +20
mV was consistently followed by a slowly activating "transient inward current" during the rise of [Ca2+]i. Further, a slowly decaying tail current was observed on repolarization of the membrane. Both the "transient inward
current" and the slowly decaying tail currents were abolished by depletion of SR Ca2+ stores by incubation of
myocytes in thapsigargin or caffeine (data not shown). A
similar (intracellular Ca2+ store-dependent) transient inward current and large slowly inactivating inward tail
currents, abolished by replacement of extracellular Na+
with Li+, were also reported in myopathic hamster myocytes overexpressing the exchanger (Hatem et al., 1994
).
Measurement of Exchanger Current in the Ca2+ Influx Mode
Normal and transgenic myocytes dialyzed with 10 mM Na+ were depolarized to +60 or +80 mV to minimize the influx of Ca2+ through the Ca2+ channel and enhance Ca2+ influx via the exchanger.
In myocytes from control mice (Fig. 8 A) a small
Ni2+-sensitive INa-Ca consistently accompanied a small
rise in [Ca2+]i in thapsigargin-treated myocytes (Fig. 8
B). Even though the rise in myoplasmic [Ca2+] induced
by the exchanger was small in control myocytes (Fig. 8
A), repolarizing to 80 mV activated ICa "tails" triggering significant release of Ca2+. In thapsigargin-treated
myocytes, the rise in [Ca2+]i in response to depolarization was somewhat larger than in control myocytes (Fig.
8 B), but Ca2+ release triggered by Ca2+ channel tail
current was absent (compare Fig. 8 A with B). These results suggest that the small rise of [Ca2+]i induced by
the influx of Ca2+ via the exchanger in control mice is
blunted by the SR activity. In transgenic myocytes, however, depolarizing pulses to less positive voltages (+60
mV, Fig. 8, C and D) produced much larger rises in
[Ca2+]i, often triggering Ca2+ release from the SR (Fig.
8 C) which activated a transient current in the inward
direction at +60 mV, representing Ca2+ extrusion by
the exchanger (Fig. 8 C). 6-8 min exposure of transgenic myocytes to 1.0 µM thapsigargin completely suppressed
Ca2+ release and the inwardly directed transient current deflections (Fig. 8 D). Instead, intracellular Ca2+
slowly but continuously increased to values of 500-600
nM during the depolarizing pulses. 5 mM Ni2+ blocked
INa-Ca, completely suppressed the rise in intracellular Ca2+, and abolished the slowly decaying exchanger-generated tail currents following the repolarization
(Fig. 8 D).
Thus, in sharp contrast to myocytes from control mice, the transgenic myocytes produce large and rapid rises in myoplasmic Ca2+ in response to activation of the Na+-Ca2+ exchanger. In 9 of 26 transgenic myocytes, the influx of Ca2+ via the Na+-Ca2+ exchanger caused rapid rise in myoplasmic Ca2+ (SR Ca2+ release) at +60 to +80 mV, the rate of which was strongly suppressed by caffeine. In 11 myocytes significant rise of [Ca2+]i was observed by depolarization without triggering Ca2+ release. In the remaining 6 myocytes, the large depolarizations failed to cause significant rise of [Ca2+]i even though large caffeine-induced INa-Ca was recorded. In contrast to transgenic myocytes, in only 5 of 15 control myocytes, significant rise of [Ca2+]i was observed upon application of large and long depolarizations. In the other 10 myocytes no significant change of [Ca2+]i could be observed at all, and in none of the 15 cells could we trigger Ca2+ release from the SR even at +80 mV.
Fig. 9 shows that Ca2+ influx via the exchanger is significantly blunted by a functional SR even in transgenic
mice. In this myocyte, depolarization from 80 to 80 mV caused only slight increase in [Ca2+]i, although repolarization from 80 to
80 mV triggered Ca2+ release
via Ca2+-influx through the deactivating L-type Ca2+
channels. Ni2+ at 5 mM concentration blocked both
Ca2+ influx transported via the exchanger and the Ca2+
channels. The same myocyte, treated with 5 mM caffeine however, showed significant rise in [Ca2+]i during
the pulse to +80 mV (Fig. 9 B). This large rise of [Ca2+]i
was completely suppressed by 5 mM Ni2+.
Relative Contributions of the Na+-Ca2+ Exchanger and Ca2+ Channel in Signaling of Ca2+ Release in Transgenic Mice
Fig. 10 examines the ability of the exchanger and Ca2+
channel to detect local rises in [Ca2+]i in transgenic
myocytes dialyzed with 2 mM Fura 2 (high concentrations of Ca2+ buffer were used to prevent significant
rise in global cytoplasmic Ca2+ concentrations; Adachi-Akahane et al., 1996; Sham et al., 1995a
). The experiment was designed to induce Ca2+ release first by caffeine and then by ICa. Thus it was possible to measure
almost simultaneously the magnitude of INa-Ca activated by caffeine, or ICa activated by depolarization, and their
respective Cai-transients. Although 2 mM Fura-2 completely suppressed the activation of caffeine-induced
INa-Ca, it did not suppress cross signaling between DHP
and ryanodine receptors (Fig. 10 B). Fig. 10 B also
shows that ICa, activated following the depletion of Ca2+
pools by caffeine, was somewhat larger, triggered a
small Cai-transient, and inactivated significantly more
slowly (Fig. 10 C ). These results were similar to those
observed in control mice myocytes and suggest that
even though the density of the exchanger current is
strongly enhanced in transgenic mice, the exchanger
remains more susceptible to cytoplasmic Ca2+-buffering than the Ca2+ channel. Thus, in the presence of 2 mM Fura-2, the Ca2+ channel continues to signal Ca2+
release (Fig. 10 A), and is in turn regulated by Ca2+ released via the ryanodine receptors (Fig. 10 C ). We conclude that increasing the density of INa-Ca by at least
threefold does not give the exchanger the type of access to the ryanodine receptor as that of the Ca2+ channel.
Fig. 11 illustrates an experimental protocol to determine the fractional contribution of the Ca2+ channel
and the exchanger in signaling Ca2+ release from the
SR in transgenic myocytes dialyzed with low concentrations of Ca2+ buffer (0.1 mM Fura 2). Transgenic myocytes were voltage clamped at 10-s intervals, from 60
to
10 mV and from
60 to +50 mV in control solutions and following 10-20-s application of 0.1 mM Cd2+. Fig. 11 A shows that in control solution activation
of Ca2+ currents triggers Ca2+ release in transgenic myocytes and that rapid application of Cd2+ blocks ICa and
the rise in [Ca2+]i (traces marked with *). In panel B,
the cell is clamped to +50 mV activating an outward
current accompanied by a small rise in [Ca2+]i. Upon
repolarization, a slowly decaying inward INa-Ca is measured following the triggering of Ca2+ release by the
Ca2+ channel tail current. The traces marked (by *)
were obtained in the presence of 0.1 mM Cd2+ and
show that Cd2+ blocked the Ca2+ channel tail current,
the rapid rise in [Ca2+]i, and the accompanying INa-Ca.
This is similar to findings in normal mice myocytes (not
shown) and reported previously in rat heart (Sham et
al., 1992
). Thus it appears that the Ca2+ channel, even
in myocytes overexpressing the exchanger, remains the
primary pathway for gating the ryanodine receptor.
The main finding of this report is that we have succeeded in producing transgenic mice that overexpress the canine sarcolemmal Na+-Ca2+ exchanger (NCX1) specifically in heart tissue. The report provides both biochemical and functional evidence for overexpression in ventricular myocytes. The Na-Ca exchange current density in transgenic mice myocytes was three times higher, on the average, compared to that measured in control myocytes (Table II). Overexpression of the exchanger did not significantly alter the Ca2+ content of Ca2+-release pools or enhance the contribution of Na+-Ca2+ exchanger to the Ca2+ release process. Overexpression of the exchanger did, however, accelerate the rate of removal of Ca2+ from the cytosol, when SR uptake was impaired by caffeine. Even though overexpression of the exchanger failed to trigger Ca2+ release at physiological membrane potentials, the exchanger did appear to blunt the rate of Ca2+ release gated by ICa in transgenic myocytes.
Consequences of Overexpression of the Exchanger
Since the exchanger is a major pathway for Ca2+ extrusion from the cytosol, it may be expected that its overexpression would reduce the Ca2+ content of the SR.
Table II clearly shows that there was no significant decrease in Ca2+ content of SR as assessed from the magnitude of caffeine-induced Ca2+ release ([Cai], column 3). Such a finding is consistent with the absence
of significant changes in cardiovascular phenotypic parameters (e.g., heart rate and blood pressure) in transgenic mice overexpressing exchange activity (H. Rockman, personal communication). Table I, also, shows that
there were no significant differences in the density of
Ca2+ channel current in control vs. the transgenic myocytes. This finding supports the observation of a recent
report (Silverman et al., 1995
) that the duration of the
action potential in these transgenic myocytes does not
change significantly at 50% duration, a period where
ICa may be the predominant inward current. Prolongation of the action potential measured at 90% of its duration reported in the same study may have been caused
by the larger inward exchanger current (see Fig. 7).
Overexpression of the Exchanger and Ca2+ Microdomains of DHP/Ryanodine Receptors
Recent data using high concentrations of Ca2+ buffers
suggests that Ca2+ signaling in cardiac muscle occurs
via microdomains of Ca2+ (Sham et al., 1995a; Adachi-Akahane et al., 1996
). Fig. 10 shows that the release of
Ca2+ by rapid application of caffeine in transgenic myocytes failed to activate the inward exchanger current in
the presence of 2 mM Fura-2 (e.g. Fig. 6), even though
the kinetics of inactivation of ICa were markedly altered
after the release of Ca2+ from the SR. One possible interpretation is that the release of Ca2+ from the ryanodine receptor is effectively buffered by 2 mM Fura-2, placing the exchanger at microdomains outside of those
surrounding DHP and the ryanodine receptor. The differential sensitivity of the exchanger for Ca2+ transport
and the Ca2+ channel to Ca2+-induced inactivation,
however, may also contribute to the data of Fig. 10. The
sensitivity of Ca2+ sites on the two proteins, however,
suggests about 5 µM affinity for the Ca2+ transport site
of the exchanger (Matsuoka and Hilgemann, 1992
) vs.
much higher Ca2+ for Ca2+ channel inactivation (10-15
µM Haack and Rosenberg, 1994
; and 50-100 µM Morad et al., 1988
). Thus the failure to activate INa-Ca while strongly modulating the kinetics of inactivation of Ca2+
channel is more consistent with the idea that the exchanger remains excluded from the Ca2+ microdomains
surrounding the DHP/ryanodine receptor complex, even in the transgenic mice.
Physiological Role of the Exchanger in Transgenic Myocytes
One reason for developing these transgenic mice was to enhance the exchanger activity in the Ca2+-influx mode. The activity of the exchanger was enhanced in most myocytes to levels where the density of current carried by the exchanger in the Ca2+ efflux mode was about 5 pA/pF, compared to 1.6 pA/pF in control myocytes (see Table II). Assuming that a similar increase in the activity of the exchanger takes place at positive potentials (Ca2+ influx mode of the exchanger), densities of current equivalent to those of Ca2+ current may be generated by the exchanger.
Ca2+ influx via the exchanger when activated by large
and long depolarizing pulses did trigger Ca2+ release in
9 out of 26 transgenic myocytes. However, ~100-300 ms were required for the exchanger to activate the
Ca2+-induced Ca2+ release mechanism (Fig. 8). In part,
because of relatively low capacity of the exchanger versus that of the SR Ca2+ pump (which may prevent significant accumulation of cytosolic Ca2+), the exchanger
fails to trigger Ca2+ release on beat-to-beat basis, especially at high mouse heart rates (~6 Hz). In the physiological range of membrane potentials, 10 to +20 mV,
we consistently failed to produce sufficient influx of
Ca2+ via the exchanger to trigger Ca2+ release in transgenic myocytes dialyzed with 0.1 mM Fura 2 (Fig. 11),
even though Ca2+ release triggered by Ca2+ current induced a large inward exchanger current (Fig. 7). Even though the exchanger may not have direct access to
Ca2+ microdomains of the DHP/ryanodine receptor
complex even when overexpressed, the finding that
Ca2+ release in transgenic myocytes was significantly enhanced when the exchanger was blocked by Ni2+ (Table II), places the exchanger within distances close
enough to the ryanodine receptor to blunt the Ca2+-induced Ca2+-release process. Thus, the overexpressed
exchanger appears to produce functional consequences
in the Ca2+ efflux, but not in the Ca2+ influx mode.
Original version received 21 October 1996 and accepted version received 20 March 1997.
Address correspondence to Dr. Morad, Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007. Fax: 202-687-8458.
A preliminary report of this work has been previously published in abstract form (Adachi-Akahane, S., K.D. Philipson, and M. Morad. 1996. Biophys. J. 70:A270).We thank Dr. D. Nicoll for assistance with molecular biology and Dr. H. Cheroutre and K. Williams (UCLA) for producing the transgenic mice. We thank Dr. Lars Cleemann for many useful discussions with measurements of Fura-2.
This work was supported by National Institutes of Health grant HL48509 and Laubisch Fund to K. Philipson and HL16152 to M. Morad.