Download Molecular properties of cardiac tail

Am J Physiol Heart Circ Physiol 288: H1810 –H1819, 2005.
First published December 9, 2004; doi:10.1152/ajpheart.01015.2004.
Molecular properties of cardiac tail-anchored membrane protein SLMAP are
consistent with structural role in arrangement of excitation-contraction
coupling apparatus
Rosa M. Guzzo,1 Maysoon Salih,1 Edwin D. Moore,2 and Balwant S. Tuana1
1
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario; and
Department of Physiology, University of British Columbia, Vancouver, British Columbia, Canada
2
Submitted 5 October 2004; accepted in final form 2 December 2004
a specialized subcellular
architecture that is necessary for the regulation of signal
transduction underlying the process of excitation-contraction (E-C) coupling (1, 3, 4, 7, 21). The sarcoplasmic
reticulum (SR), derived from the endoplasmic reticulum
(ER), constitutes an abundant internal membrane system
comprised of two distinct membrane domains: the longitudinal (nonjunctional) SR and the terminal cisternae (junctional) SR (7, 27). Situated in close apposition to the
terminal cisternae SR is a system of sarcolemmal invaginations, known as transverse (T)-tubules. Localized entry of
calcium ions through the L-type calcium channel situated at
the T-tubules is induced on depolarization of the sarcolemma and triggers the release of calcium from the SRlocalized calcium release channels into the cytosol (3, 4, 6,
20). This increase in intracellular calcium levels activates
the closely situated contractile units composed of the myofilaments to facilitate myocyte shortening. The subsequent
removal of calcium by the activities of the SERCA pumps of
the longitudinal SR and the Na⫹/Ca2⫹ exchanger present at
the T-tubules and sarcolemma results in relaxation (12, 19,
22). Thus the proper spatial distribution of these membrane
systems relative to the myofilaments is pivotal for the
coordination of the E-C mechanism (1, 3, 4, 7).
The precise mechanisms that organize the membrane
architecture of the cardiac myocyte so that the appropriate
targeting of the E-C coupling apparatus can be achieved and
maintained on a beat-to-beat basis remain poorly understood. Thus the identification of the molecular components
necessary for the organization and stabilization of the cardiac membrane architecture would not only advance the
understanding of cardiomyocyte function but also offer
insights into heart disease where membrane disorganization
has been correlated with dysfunction (1, 8, 9). Recent
studies indicate that a family of tail-anchored membrane
proteins designated the junctophilins (JPs) contributes to the
formation of the dyadic couplings between the SR/ER and
the T-tubules in cardiomyocytes (13, 25, 26). A COOHterminal hydrophobic segment targets the JPs to the ER/SR
membrane, whereas the cytoplasmic domain interacts with
lipid moieties of the plasma membrane (25). Although the
JPs appear critical for membrane organization, studies also
suggest that other as yet unidentified molecular entities are
also necessary for the proper arrangement of the E-C coupling mechanism. In previous studies, we defined the primary structure of a family of coiled-coil, tail-anchored
sarcolemmal membrane-associated proteins termed the SLMAPs that were highly expressed in the myocardium (11,
28, 29). The genomic analysis of the SLMAP gene revealed
that several SLMAP isoforms were generated by alternative
splicing mechanisms with unique COOH-terminal hydrophobic domains. The SLMAPs share an overall structural
similarity to other tail-anchored membrane proteins such as
the syntaxins, which serve roles in membrane docking and
fusion events (16). Here, we provide evidence that SLMAPs
can localize at the level of the SL, T-tubules, and SR and be
targeted to distinct cellular membranes by their unique
COOH-terminal tails. We also provide evidence that
SLMAPs can self assemble and interact with cardiac myo-
Address for reprint requests and other correspondence: B. S. Tuana, Dept. of
Cellular and Molecular Medicine, Univ. of Ottawa, Ottawa, Ontario, Canada
K1H 8M5 (E-mail: [email protected]).
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.
sarcoplasmic reticulum; transmembrane domain; sarcolemmal membrane-associated protein
THE CARDIOMYOCYTE HAS EVOLVED
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0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society
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Guzzo, Rosa M., Maysoon Salih, Edwin D. Moore, and
Balwant S. Tuana. Molecular properties of cardiac tail-anchored
membrane protein SLMAP are consistent with structural role in
arrangement of excitation-contraction coupling apparatus. Am J
Physiol Heart Circ Physiol 288: H1810 –H1819, 2005. First published December 9, 2004; doi:10.1152/ajpheart.01015.2004.—The
spatial arrangement of the cell-surface membranes (sarcolemma and
transverse tubules) and internal membranes of the sarcoplasmic reticulum relative to the myofibril is critical for effective excitationcontraction (E-C) coupling in cardiac myocytes; however, the molecular determinants of this order remain to be defined. Here, we ascribe
molecular and cellular properties to the coiled-coil, tail-anchored
sarcolemmal membrane-associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of
the cardiomyocyte. The expression of SLMAP was developmentally
regulated and its localization was distinctly apparent at the level of the
membranes involved in regulating the E-C coupling mechanism.
Several SLMAP isoforms were expressed in the cardiac myocyte with
unique COOH-terminal membrane anchors that could target this
molecule to distinct subcellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in
cardiac muscle. The cardiac-specific expression of SLMAP isoforms
that can be targeted to distinct subcellular membranes, self assemble,
and interact with the myofibril suggests a potential role for this
molecule in the structural arrangement of the E-C coupling apparatus.
MOLECULAR PROPERTIES OF SLMAP
sin. These properties imply a role for these molecules in the
structural arrangement of the E-C coupling mechanism of
the cardiomyocyte.
MATERIALS AND METHODS
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beads with constant rotation at 4°C. Beads were separated from the
lysate by brief centrifugation (4,000 rpm, 10 s), and the protein-linked
beads were washed several times with lysis buffer. Two additional
washes were performed with purification buffer (PBS, pH 7.4, 0.5%
NP-40, 0.8% NaCl, 5% glycerol).
Glutathione S-transferase pull-down assays. H9c2 cells were lysed
in RIPA buffer supplemented with protease inhibitor cocktail (Sigma),
and unbroken cells were removed by centrifugation. The H9c2 cell
lysate was precleared with glutathione Sepharose 4B (Amersham
Pharmacia) beads linked to glutathione S-transferase (GST; 30-min
rotation at 4°C). Six milligrams of precleared cell lysate were incubated overnight at 4°C with equivalent amounts of purified GST or
GST-SLMAP1 bound to glutathione Sepharose 4B beads. After being
bound, the beads were extensively washed with 1⫻ RIPA, 0.5⫻ RIPA
or PBS, and then resuspended in 2⫻ sample buffer. Captured proteins
were eluted from the beads by boiling and separated on 7.5% SDSPAGE gels.
Microsomes were isolated from adult rabbit ventricles by homogenization in buffer consisting of 50 mM Tris 䡠 HCl, pH 7.8, 20 mM
sodium tetraphosphate, and 1 mM EDTA supplemented with protease
inhibitor cocktail (Sigma). The homogenate was cleared of debris by
centrifugation at 10,000 g for 10 min at 4°C and then filtered through
cheesecloth. KCl was added to a final concentration of 0.6 M, and the
cleared homogenate was further centrifuged at 100,000 g for 45 min
at 4°C. The pellet was retained and solubilized overnight with constant rotation in buffer consisting of 12.5 mM MOPS, pH 7.2, 0.8%
CHAPS, 0.5% NP-40, 450 mM NaCl, 150 mM sucrose, and 25 ␮M
EGTA. Solubilized membranes were then subjected to high-speed
centrifugation (190,000 g, 1 h). The supernatant was retained as the
solubilized crude microsome fraction.
Silver staining, digestion, and mass spectrometry. SDS-polyacrylamide gels were fixed in 50% ethanol, 5% acetic acid solution for 30
min at room temperature. Gels were then washed for 10 min in 50%
ethanol followed by two 10-min washes in double-distilled water and
incubated in silver-staining sensitizer solution (0.02% sodium thiosulphate) for 5 min and then washed twice in double-distilled water
before staining in 0.1% silver nitrate for 30 min. Gels were briefly
rinsed in double-distilled water and then exposed to developer solution (0.04% formalin in 2% sodium carbonate). This step was carefully monitored, and the reaction was stopped by the addition of 5%
acetic acid solution on visualization of bands. Gels were stored in 1%
acetic acid solution. Gel slices were destained (15 mM potassium
ferricyanide, 50 mM sodium thiosulphate) and incubated at room
temperature with shaking. Gel pieces were then washed (3 ⫻ 10 min)
in deionized water, and acetonitrile was added to shrink the gel pieces.
Gel pieces were rehydrated with buffer consisting of 0.01 ␮g/␮l of
trypsin in 50 mM ammonium bicarbonate and digested overnight at
37°C. The buffer containing the digested peptides was transferred into
a mass spectrometer analysis plate and analyzed on the Q-tof ultima
instrument (Micro Mass, courtesy of Dr. J. Kelly, National Research
Council of Canada). Samples were analyzed using a nano-LC-MS/MS
method with runs of 45 min/samples and a gradient of acetonitrile
ranging from 5 to 55%.
Cell culture and transfections. COS7 African green monkey kidney
cells and H9c2 cells (CRL-1446, ATCC) were maintained at 37°C in
DMEM supplemented with 10% heat-inactivated FBS and antibiotics.
Transient transfection experiments were performed using the Fugene
(Roche Biochemicals) transfection reagent according to the manufacturer’s specifications. 6Myc-tagged SLMAP1 expression constructs
encoding transmembrane domain 1 (TM1), TM domain 2 (TM2), or
the construct lacking both TM (⌬TM) domains were generated.
Immunocytochemistry. Cells grown on sterile glass coverslips were
fixed in either 4% paraformaldehyde in phosphate buffer for 15 min at
room temperature or in ice-cold methanol for 5 min. After either
fixation method, coverslips were mounted onto glass slides and cells
were incubated with the relevant antibody(s) diluted in 0.01 M PBS
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Immunohistochemistry. Timed pregnant mice were killed by lethal
injection of Somital. Embryos (9 –15 days postcoitum) were removed,
rinsed briefly in PBS, and then immediately fixed in 4% paraformaldehyde in 100 mM phosphate buffer, pH 7.4. For cryoprotection,
embryos were incubated in 20% sucrose, followed by incubation in
15% sucrose in PBS. Fixed embryos were embedded in Tissue-Tek
O.C.T. compound, frozen, and stored at ⫺80°C. Cryosections (6 –10
␮m) were collected on gelatin-coated microscope slides and stored at
⫺80°C. Older embryos (18 days postcoitum) and adult tissues were
isolated, rinsed in PBS, embedded in Tissue-Tek OCT compound, and
then frozen. Cryosections were mounted onto slides and fixed overnight in 4% paraformaldehyde. Immunohistochemistry was performed
by first prewarming slides to 37°C. Paraformaldehyde-fixed sections
were washed in PBS and then treated with 50 mM ammonium
chloride in PBS for 5 min to reduce nonspecific staining of blood
proteins. Sections were blocked in PBS containing 10% goat serum,
1% Triton X-100, and 10% bovine serum albumin for 20 min at room
temperature before being exposed to primary antibodies (1 h at room
temperature). After several washes in PBS, sections were incubated in
PBS containing 5% goat serum, 1% Triton X-100, and the appropriate
fluorochrome-linked secondary antibodies for 1 h at room temperature. After several washes in PBS, sections were mounted with
antifading solution (Molecular Probes).
Antibodies. The following antibodies were used in the immunocytochemistry/histochemistry studies: monoclonal anti-Myc 9E10
monoclonal antibody (Dr. J. Bell, University of Ottawa); anti-␣actinin monocolonal antibody (Sigma, clone BM-75.2); monoclonal
anti-caveolin-3 antibody, anti-ryanodine receptor (Transduction Laboratories). Anti-SLMAP(C) rabbit antisera was raised against the
COOH 370 amino acids of SLAP, as previously described (29). The
secondary antibodies used in these studies included Alexa-Fluor
488-conjugated anti-rabbit immunoglobulins and Alexa Fluor 594conjugated anti-mouse immunoglobulins (Molecular Probes).
Generation of glutathione S-transferase fusion proteins. Rabbit
SLMAP1 cDNA was PCR amplified from full-length SLMAP3
cDNA using the forward primer (GEXSL15⬘: cgGAATTCaaagcagtgacgacac) and reverse primer (GEX3⬘: cgGAATTCgtgtacggactcaagaaa). Full-length SLMAP3M1 cDNA was generated by PCR using
the GEXM15⬘ forward primer (cgGAATTCccagtctgtcctcgatg) and
GEX3⬘ reverse primer (cgGAATTCgtgtacggactcaagaaa). PCR conditions consisted of 1) 3 min at 94°C; 2) 30 cycles of 94°C for 40 s
(denaturing), 60°C for 30 s (annealing), 72°C for 2.5 min (extension);
followed by 3) one last step at 72°C for 10 min. PCR products were
EcoRI digested and ligated into EcoRI site of pGEX-2TK vector
(source) to yield pGEX-SLMAP1. Construct orientation was verified
by restriction site mapping and sequencing. pGEX-2TK or pGEXSLMAP1 was used to transform BL21 Codon Plus (Stratagene)
competent cells. Transformed cells were plated on LB agar plates
supplemented with ampicillin (1,000 ␮g/ml), chloramphenicol (40
␮g/ml), and 2% glucose. Overnight bacterial cultures for each transformant were grown at 37°C until an optical density reading (A600nm)
of 0.5– 0.6 was reached. Fusion protein synthesis was induced with
0.1 mM IPTG for 4 h at room temperature. Cells were collected by
centrifugation for 10 min at 5,000 rpm and resuspended in lysis buffer
(PBS, pH 7.4, 1% Triton X-100, 0.8% NaCl, 5% glycerol) supplemented with protease inhibitor cocktail (Sigma). To lyse the cells, 0.1
mg/ml of lysozyme was added and lysates were incubated on ice for
30 min, followed by brief sonication. Sonicated material was centrifuged for 20 min at 20,000 g, and collected supernatants were then
incubated with glutathione Sepharose 4B (Amersham Pharmacia)
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MOLECULAR PROPERTIES OF SLMAP
RESULTS
SLMAP expression in the developing myocardium and adult
cardiomyocytes. Our previous studies indicate that there is
robust SLMAP mRNA and protein expression in adult cardiac
muscle (28, 29), but nothing is known about its expression in
the developing myocardium. At 9 days postcoitum, the primitive heart exists as a tubular structure with presumptive atrial
and ventricular chambers. SLMAP-antibody labeling of formalin-fixed sagital cryosections from whole mouse embryos reAJP-Heart Circ Physiol • VOL
vealed that SLMAP was expressed in both the atria and
ventricles at this early developmental stage; however, a more
robust staining was observed in the ventricular myocardium
(Fig. 1A). At 13 days postcoitum, the developing heart is
divided by septa into a four-chambered heart and SLMAP was
found to be uniformly expressed in the atrial and ventricular
myocardium, as well as the interventricular and interatrial
septum (Fig. 1B, a, c, d). No immunostaining was observed in
sections incubated in preimmune rabbit serum (Fig. 1Bc) or in
the absence of the primary antibody (not shown). Confocal
images of ventricular myocytes stained for anti-SLMAP at 13
days postcoitum revealed diffuse SLMAP localization, and a
distinct distribution of SLMAPs relative to the myofibrils was
not observed at this stage of development (Fig. 2, a-c). In older
embryos (18 days postcoitum), a clear striated pattern of
SLMAP localization was apparent and mostly distinct from
that of the Z-line marker ␣-actinin (Fig. 2, d-f). Immunostaining and confocal microscopy were used to further examine the
subcellular localization of SLMAP in isolated adult rat ventricular myocytes. A clear cross-striated pattern of SLMAP
distribution was observed in adult cardiac cells (Fig. 2, g-i).
Anti-SLMAP labeling of the Z-line was apparent by confocal
microscopy (Fig. 2i); however, substantial SLMAP staining
was also noted at reticular structures perpendicular to the
Z-line staining. Further analysis of SLMAP localization in
single adult cardiomyocytes using confocal and deconvolution
techniques (Figs. 3 and 4) indicated that SLMAP-specific
staining extended toward the periphery of the cell as well as the
invaginations of the plasma membrane (Fig. 3). Thus we
examined whether SLMAP codistributed with markers of external membrane systems and the internal membranes such as
the SR. Caveolin-3 proteins are expressed in cardiac, skeletal,
and smooth muscle and reside at the vesicular invaginations of
the plasma membrane known as caveolae (24). Caveolin-3
antibodies clearly labeled a subcompartment of the surface
cardiac membrane as well as plasma membrane invaginations
(Figs. 3b and 4B), consistent with the distribution of caveolin-3
in the T tubular membrane system in cardiac cells (17).
SLMAP was seen to colocalize with caveolin-3 at the cellsurface membrane invaginations and along the Z-lines (white
voxels) as resolved by deconvolution microscopy (Figs. 4B and
3c). Regional differences in the SLMAP-caveolin-3 colocalization were apparent as caveolin-3 along the Z-lines was more
heavily colocalized with SLMAP (white voxels) than the cell
surface (Figs. 3c and 4B).
The ryanodine receptors (RyRs) or the calcium release
channels are essential components of the E-C coupling machinery and are localized at the terminal cisternae SR membranes. By immunofluorescent microscopy, RyRs are typically
detected as a series of regularly spaced doublets along the
Z-disc in the cardiomyocytes (Figs. 3e and 4C in red) (8). Dual
staining with SLMAP and RyR2-specific antibodies revealed
substantial colocalization of these molecules along the Z-disc
as indicated by white voxels (Figs. 3, d-f, and 4C). Thus Fig. 4
shows that the SLMAP is distributed in multiple locations but
it is prominent at the cell surface around the nuclear envelope
and along the Z-lines. At the cell surface and in the nuclear
envelope 32 ⫾ 6% of the voxels containing signal specific for
caveolin-3 also contained SLMAP (n ⫽ 3; Fig. 4B). Although
at the Z-lines, 36 ⫾ 7% (n ⫽ 4) of the voxels containing the
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containing 0.3% Triton X-100 (PBS-T) for 2 h at room temperature.
After several washes in PBS, cells were incubated in the appropriate
fluorochrome-conjugated secondary antibody(s) for 45 min at 37°C.
Cells were washed extensively in PBS and then mounted with antifade
media (Molecular Probes).
Cell isolation and labeling. Ventricular myocytes were isolated
from adult male Wistar rats and fixed using methods previously
described (23). The fixed cells were then adhered to acid-washed
coverslips using poly-L-lysine (Sigma), rinsed with 3 ⫻ 10-min
washes in PBS (in mM: 137 NaCl, 8 NaH2PO4, 2.7 KCl, 1.5 K
H2PO4, pH 7.4), permeabilized in a PBS solution containing 0.1%
Triton X-100 for 10 min, and given a final 3 ⫻ 10-min rinse in PBS.
The cells were then incubated simultaneously with the polyclonal
anti-SLMAP (1:100) and a monoclonal antibody, either anti-caveolin-3 (1 ␮g/ml, BD Transduction Laboratories) or anti-ryanodine
receptor (1 ␮g/ml, Affinity Bioreagents) overnight in a humidified
environment at room temperature. The antibodies were diluted in
antibody buffer (in mM: 75 NaCl, 18 Na3citrate, 2% goat serum, 1%
BSA, 0.05% Triton X-100, and 0.02% NaN3). After 3 ⫻ 10-min
rinses in antibody wash solution (in mM: 75 NaCl, 18 Na3citrate,
0.05% Triton X-100), the cells were incubated with highly crossadsorbed anti-rabbit and anti-mouse secondary antibodies that were
labeled with fluorescein isothiocyanate (FITC) or Texas red (Jackson
Immunobiologicals) for 2 h at room temperature and then given a final
3 ⫻ 10-min rinse in antibody wash solution. Some of the coverslips
were used for control experiments and were incubated only with the
antibody buffer overnight and then labeled with the fluorescently
tagged secondary antibodies. All of the coverslips were mounted onto
frosted slides in a solution composed of 90% glycerol, 10% 10⫻ PBS,
2.5% triethylenediamine, and 0.02% NaN3. Fluorescent microspheres
(0.2-␮m diameter, Molecular Probes) labeled with FITC and Texas
red were added to the mounting medium to act as fiduciary markers
and permit accurate alignment of the three-dimensional data sets.
Image acquisition and deconvolution. A series of two-dimensional
images were acquired through the cells at 0.25-␮m spacing using a
Nikon Diaphot 200 microscope and a Planapo 60/1.4 objective. This
configuration produced voxel dimensions of 100 ⫻ 100 ⫻ 250 nm and
satisfied the Nyquist criteria for sampling and prevented aliasing. The
image detector was a thermoelectrically cooled CCD camera with a
Site S1502AB chip with a peak quantum efficiency of 80%. Other
details of the microscope can be found in Scriven et al. (23). The point
spread function of the microscope was measured using fluospheres of
the appropriate color (0.1-␮m diameter, Molecular Probes). The
images were prepared, deconvolved, and analyzed as previously
reported (5).
Microscopy. Samples were visualized using Axiophot (Carl Zeiss)
microscope equipped with a 3CCD color video camera. An Olympus
IX70 laser-scanning inverted microscope with a ⫻63 oil immersion
objective was also used for observation. Images were acquired using
the Bio-Rad MRC 1024 confocal. Acquired images were digitally
processed using Northern Eclipse (Version 5.0, Empix Imaging)
acquisition software as well as the Confocal Assistant 40 software.
Images were further processed using Adobe Photoshop 5.0 (Adobe
Systems).
MOLECULAR PROPERTIES OF SLMAP
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RyR also contained SLMAP (Fig. 4C). Each of the images also
shows prominent staining of the M-line by the SLMAP antibody.
Unique COOH-terminal anchors in SLMAP target distinct
cellular membranes. Our previous studies indicate that three
distinct mRNAs encoding SLMAP polypeptides of 35, 60, and
⬃80 kDa are expressed in cardiac muscle, and biochemical
fractionation experiments revealed that these polypeptides are
enriched in surface membrane and SR fractions (17). Our
previous studies also reveal that alternative splicing generates
SLMAPs with unique COOH-terminal TM domains TM1 or
TM2 that are equally expressed in the myocardium (18).
Whether these unique COOH-terminal membrane anchors can
target SLMAPs to distinct subcellular membranes is not
known. We therefore examined whether the unique COOHterminal TM domains in SLMAPs can target these polypeptides to distinct subcellular organelles. SLMAP constructs
encoding either TM1 or TM2 sequences were fused to the
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6Myc epitope tag and transfected into COS7 cells and examined by immunohistochemistry. Transient expression of
SLMAP sequences encompassing TM1 generated a pronounced perinuclear distribution, which also extended throughout the cytoplasm in a diffuse reticular pattern consistent with
the distribution of the ER (Fig. 5A). Immunostaining with
anti-calnexin confirmed the identity of the SLMAP-localized
compartment as ER (data not shown).
Ectopic expression of TM2 sequences targeted the 6Myctagged SLMAP fusion protein to distinct reticular formations
as well as tubular-like projections throughout the cell that were
not observed in cells expressing TM1 (Fig. 5A). Extraction of
the cells with Triton X-100 before fixation resulted in lack of
myc staining in paraformaldehyde-fixed cells expressing
6Myc-SLMAP constructs, indicating that 6Myc-SLMAP is
targeted to subcellular membrane structures (data not shown).
A characteristic feature of all cardiac SLMAP isoforms is the
presence of a coiled-coil leucine-rich motif in the COOH-
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Fig. 1. Sarcolemmal membrane-associated protein (SLMAP)
expression in the developing myocardium. A: sagital sections (6
␮m) of formalin-fixed mouse embryos were eosin-hematoxlyin
stained (a and b) or subject to immunohistochemical analysis
using anti-SLMAP rabbit polyclonal antibodies (c and d). At
embryonic day 9, SLMAP-specific labeling (c and d) was
observed within both the presumptive atrial (A) and ventricular
(V) chambers, with greater expression observed in the ventricle.
Scale bar ⫽ 250 ␮m. B: at 13 days postcoitum, SLMAPs were
uniformly expressed in the A and V myocardium, as well as the
interventricular septum (IVS) and interatrial septum (a, c, and
d). An immunofluorescent signal was not observed within the
myocardium of 13-day postcoitum hearts using preimmune
rabbit serum in sequential (transverse) sections (b).
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MOLECULAR PROPERTIES OF SLMAP
terminal region encoded by exons 20 and 21. When the
leucine-rich coiled-coil region was deleted from SLMAP, it
still targeted the reticular structures (Fig. 5Ac), but when the
TM domain was deleted, SLMAP was seen to be diffusely
distributed throughout the cytoplasm (Fig. 5Ad). To further
examine whether the expression of the two distinct COOHterminal TM anchors mediates differential SLMAP-membrane
associations, cotransfection experiments were performed using
1) SLMAP cDNA encompassing TM1 fused to 6Myc (6MycSLMAP-TM1) and 2) SLMAP cDNA encompassing TM2
fused to green fluorescent protein (GFP-SLMAP-TM2). The
subcellular distribution of the 6Myc-SLMAP-TM1 fusion protein as well as the GFP-SLMAP-TM2 fusion proteins was
AJP-Heart Circ Physiol • VOL
monitored by visualizing GFP immunofluorescence relative to
myc antibody labeling in cells coexpressing the two SLMAP
fusion proteins (Fig. 5B). Whereas some overlap in the GFPmyc immunostaining was apparent in cells coexpressing TM1
and TM2, the localizations were predominantly distinct (Fig.
5B). Inclusion of the GFP tag did not appear to affect membrane targeting as cells that were cotransfected with GFPSLMAP and 6Myc-SLMAP fusion proteins encompassing the
same membrane anchor (TM2) showed completely overlapping distribution profiles (Fig. 5B). Thus it appears that the
function of the two different membrane anchors in SLMAP is
to target this polypeptide to distinct subcellular membrane
compartments.
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Fig. 2. SLMAP localization relative to the myofibril in developing and adult ventricular myocytes. Ventricular myocytes from 13 days postcoitum mouse
embryos (a– c), 18 days postcoitum mouse embryos (d–f ), and adult rats (g–i) were stained for SLMAP (a, d, and g) and the myofibril marker protein ␣-actinin
(b, e, and h). The merge of the SLMAP and the ␣-actinin proteins was visualized by confocal imaging at striated formations demarcating the Z-line of the
myofibril in ventricular myocytes at 13 days postcoitum (b). SLMAP-specific labeling was displayed throughout the myocytes at this stage (a). SLMAP
antibodies stained reticular formations (d) in older embryos (18 days postcoitum) and showed limited overlap within the Z-line ␣-actinin (e and f ). In isolated
adult rat ventricular myocytes costained for SLMAP (g) and ␣-actinin (h), immunoconfocal images showed areas of colocalization (yellow) (i) as well as distinct
SLMAP labeling at structures perpendicular to the ␣-actinin-labeled striations. Scale bar ⫽ 10 ␮m.
MOLECULAR PROPERTIES OF SLMAP
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SLMAP can homodimerize in vivo and bind cardiac myosin.
To gain insight into the molecular interactions of cardiac
SLMAP, GST-SLMAP fusion proteins were immobilized on
glutathione-Sepharose beads and incubated with detergent extracts of rabbit ventricles H9c2 cardiomyoblasts. After being
washed extensively to remove nonspecific and weakly bound
proteins, the specifically bound proteins were eluted and analyzed by SDS-PAGE and visualized by silver staining (Fig. 6).
Two polypeptides of apparent molecular mass ⬃35 and ⬃70
kDa were found to consistently bind to GST-SLMAP1 from
Fig. 4. Colocalization of SLMAP with caveloin-3 and RyRs. Deconvolution microscopy was used to examine cells stained with SLMAP (green, A-C), caveolin-3
(red, B), and the RyR (red, C). Colocalized voxels are white. The arrows point to the surface and to a Z-line, and the double-headed arrows point to the M-line.
n, Nucleus. Each image is 0.5 ␮m deep and the scale bar ⫽ 2 ␮m. Pixel sizes are 100 nm in x and y and there is a 250-nm spacing between image planes.
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Fig. 3. SLMAP distribution in the adult ventricular myocytes. Caveolin-3 antibodies labeled peripheral structures representing a subdomain of the plasma
membrane (b). SLMAP antibodies (a) primarily labeled internal structures. Overlay of the caveolin-3 and SLMAP staining showed that the 2 immunofluorescent
signals coincided at membrane invaginations (c). Consistent with the distribution of membrane components of the terminal cisternae sarcoplasmic reticulum, the
monoclonal anti-ryanodine receptor (RyR2) antibody labeled regularly spaced doublets (e). SLMAP antibodies (d) stained similar structures as the RyR2 antibody
as well as tubular-like structures that were distinct from RyR2 labeling (f ). Scale bar ⫽ 10 ␮m.
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Fig. 5. Distinct membrane targeting mediated by alternative SLMAP transmembrane
(TM) domains. Sequences encoding the
6Myc-epitope tag were fused in frame with
NH2-terminal sequences of SLMAP1 encompassing TM1 (6Myc-SLMAP-TM1);
SLMAP1 sequences encompassing TM2
(6Myc-SLMAP-TM2); SLMAP1 sequences
lacking either TM (6Myc-SLMAP1TM); sequences encoding the green fluorescent protein (GFP) were also fused in frame with
NH2-terminal sequences of SLMAP1 encompassing TM2 (GFP-SLMAP1-TM2).
These constructs were used in transient
transfection studies in COS7 cells. A: transient expression of the 6Myc-SLMAP1-TM1
expression construct revealed targeting of
the 6Myc heterologous protein to juxtanuclear sites and reticular formations extending throughout the cytoplasm (a). The
6Myc-SLMAP1-TMD2 fusion protein was
ectopically expressed at reticular formation
and distinct tubular structures (b). A similar
localization pattern was observed in cells
expressing 6Myc-SLMAP1-LZTMD2 (c). In
the absence of either TM domain (6MycSLMAP1TM), the Myc labeling was diffusely distributed throughout the cell and
membrane associations were not apparent
(d). Scale bar ⫽ 10 ␮m. B: cells were cotransfected with 6Myc-SLMAP1-TM1 and
GFP-SLMAP-TM2 to confirm that the 2 TM
domains did not target SLMAPs to the same
intracellular membrane sites. Cells cotransfected with 6Myc-SLMAP-TM2 and GFPSLMAP-TM2 showed complete overlap of
the Myc and GFP signals. Scale bar ⫽ 10
␮m.
cardiac muscle and cell extracts (Fig. 6). In addition, a polypeptide
of ⬃200 kDa was also eluted from the immobilized GSTSLMAP1 incubated with the cardiac fractions. Each of these
polypeptides specifically interacted with the GST-SLMAP fusion proteins, as none of these were bound by GST alone or in
other control experiments where GST or GST-SLMAP recombinant proteins were incubated with lysis buffer alone. The
⬃200-, ⬃70-, and ⬃35-kDa proteins were excised from the
AJP-Heart Circ Physiol • VOL
silver-stained gels, trypsin digested, and further analyzed for
amino acid composition by mass spectrometry. Protein database comparisons of the tryptic peptide sequences corresponding to the 70- and 35-kDa polypeptides matched accession
numbers 790240, 790238, and 20870858, each representing
SLMAP sequences. The peptide sequences acquired from the
mass spectrometric analysis of the 200-kDa protein identified with
the ␣- and ␤-subunits of myosin heavy chain of the myocardium.
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MOLECULAR PROPERTIES OF SLMAP
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We further analyzed the localization of SLMAP to see
whether it resides at the level of the M-line where myosin is
located in cardiomyocytes (Fig. 7). Images acquired from three
different rat ventricular myocytes stained with anti-SLMAP
demonstrate that SLMAP is distributed at the Z-lines (singleheaded arrows) and along the M-lines (double-headed arrows)
in cardiomyocytes.
DISCUSSION
The data here show that SLMAPs can reside at the level of
the E-C coupling apparatus of the cardiomyocytes as well as
perinuclear membrane structures including the ER. Molecular
analysis of the SLMAP gene has predicted the presence of a
large number of isoforms generated by alternative splicing
mechanisms that are expressed in a tissue-specific manner (10,
11, 28, 29). The different locations of SLMAP noted here may
reflect the presence of these diverse isoforms in cardiomyocytes. SLMAPs may potentially serve a role in the subcellular
architecture of the E-C coupling mechanism by perhaps linking
the SR to the myofibril on the one hand and organizing the
cell-surface membrane to SR junctions on the other. Immunoconfocal imaging revealed that SLMAPs are expressed early in
the developing myocardium and are localized within the SR
and cell-surface membranes of the ventricular myocytes. Confocal imaging of SLMAP expression in mature cardiomyocytes
indicates that SLMAPs are colocalized with markers of the
surface membrane such as caveolin and those of the junctional
SR such as the RyR. These immunohistochemical data are
consistent with our previous biochemical studies, which indicated that three SLMAP polypeptides of ⬃80, 70, and 35 kDa
Fig. 7. Immunohistochemistry of SLMAP at the M-line and Z-disc in cardiomyocytes. Single image planes acquired from a different rat ventricular myocyte
fixed and labeled with anti-SLMAP are shown. The single-headed arrows point to Z-lines, and the double-headed arrows point to M-lines. Scale bar ⫽ 5 ␮m.
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Fig. 6. Identification of the SLMAP-binding
partners. A: Coommassie-stained gel shows
the purified glutathione-S-transferase (GST;
26 kDa) and GST-SLMAP1 fusion protein
(GST-SLMAP1; 60 kDa) used in the “pulldown” assays. B: electrophoretic profile of
proteins isolated from the H9c2 cell lysate
that associated with GST or GST-SLP1 proteins immobilized on glutathione-Sepharose
beads. A 35- and 70-kDa protein specifically
bound the GST-SLMAP1 proteins. These
bands were not recovered in lysates incubated with GST alone or in controls consisting of buffer incubated with GST or GSTSLMAP1. C: electrophoretic profile of proteins
recovered from a rabbit heart microsomal
lysate that associated with GST or GSTSLMAP1 proteins. A 220-kDa protein specifically associated with GST-SLMAP1.
This protein did not interact with GST alone,
nor were they identified by incubating buffer
with GST or GST-SLMAP1.
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MOLECULAR PROPERTIES OF SLMAP
AJP-Heart Circ Physiol • VOL
structure in excitable cells (14 –16). Additional studies have
suggested that other molecules such as Mitsugumin 29 are also
required for effective junctional couplings. Mitsugumin 29 is a
synaptophysin family-related protein that is found to be located
in the junction between the plasma membrane and SR of
skeletal muscle and appears to contribute to the membrane
arrangement and E-C coupling (18). Our data here suggest that
the cardiac tail-anchored membrane protein SLMAP is a novel
component and perhaps a unique candidate for a role in
organizing the E-C coupling apparatus of the cardiomyocyte. In
view of the extensive alternative splicing mechanisms that generate a multitude of SLMAP isoforms, the presence of this
molecule at distinct subcellular locations suggests diverse roles in
cell function (10).
GRANTS
This work was supported by a grant to B. S. Tuana from Heart and Stroke
Foundation of Ontario, and R. M. Guzzo was supported by a studentship award
from Canadian Institutes of Health Research.
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