1 Department of Biochemistry 194, University Medical Center, NCMLS, 6500 HB
Nijmegen, The Netherlands
2 Department of Medical Biochemistry and Microbiology, Uppsala University, S-751
23 Uppsala, Sweden
3 Department of Biochemistry 160/Microscopical Imaging Center, University
Medical Center, NCMLS, 6500 HB Nijmegen, The Netherlands
* Present address: Biological Engineering Division, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA
Author for correspondence (e-mail:
a.vankuppevelt{at}ncmls.kun.nl)
Accepted 28 February 2003
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Summary |
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Immunostaining for specific heparan sulfate epitopes showed major changes in the heparan sulfate composition in skeletal muscle tissue derived from NDST-1/ mice and NDST/ cultured myotubes. Biochemical analysis indicates a relative decrease in both N-sulfation and 2-O-sulfation of skeletal muscle heparan sulfate. The core protein of heparan sulfate proteoglycan perlecan was not affected, as judged by immunohistochemistry. Also, acetylcholine receptor clustering and the occurrence of other ion channels involved in excitation-contraction coupling were not altered. In NDST-2/ mice and heterozygous mice no changes in heparan sulfate composition were observed. Using high-speed UV confocal laser scanning microscopy, aberrant Ca2+ kinetics were observed in NDST-1/ myotubes, but not in NDST-2/ or heterozygous myotubes. Electrically induced Ca2+ spikes had significantly lower amplitudes, and a reduced removal rate of cytosolic Ca2+, indicating the importance of heparan sulfate in muscle Ca2+ kinetics.
Key words: Acetylcholine receptor, Ca2+, Heparan sulfate, Sulfotransferase, Skeletal muscle
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Introduction |
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After formation of a HS precursor polysaccharide, consisting of repeating
D-glucuronic acid and N-acetyl-D-glucosamine residues, the
bifunctional enzyme glucosaminyl
N-deacetylase/N-sulfotransferase (NDST) catalyzes the first
modifications of the precursor polysaccharide, where the acetyl group of
selected N-acetyl-D-glucosamine residues is replaced with a sulfate
group. This reaction is a prerequisite for all other modifications such as C-5
epimerization, 2-O-sulfation, 3-O-sulfation, and
6-O-sulfation, which only occur in the vicinity of N-sulfate
groups (Lindahl et al.,
1998).
Four isoforms of NDST have been identified
(Hashimoto et al., 1992;
Eriksson et al., 1994
;
Orellana et al, 1994
;
Aikawa and Esko, 1999
). NDST-1
and NDST-2 are widely distributed
(Humphries et al., 1997
;
Humphries et al., 1998
;
Kusche-Gullberg et al., 1998
;
Aikawa et al., 2001
), whereas
NDST-3 and NDST-4 have a more restricted expression. Disruption of the genes
encoding NDST-1 (Fan et al.,
2000
; Ringvall et al.,
2000
) and NDST-2 (Forsberg et
al., 1999
; Humphries et al.,
1999
) gave insights in the roles of these enzymes in the
biosynthesis and physiological roles of HS and heparin, a highly sulfated form
of HS. NDST-1-deficient mice are capable of synthesizing N-sulfated
HS, but the sulfate content is significantly lower than that of HS from
wild-type mice, indicating the importance of this enzyme in HS biosynthesis
(Ringvall et al., 2000
). So
far, no obvious defects in HS have been found in NDST-2-deficient mice, which
instead are unable to synthesize sulfated heparin
(Humphries et al., 1999
;
Forsberg et al., 1999
).
However, the increased lethality of NDST-1 and NDST-2 double knockout mice,
compared to NDST-1-deficient mice, suggests that NDST-2 also plays a role in
HS biosynthesis (Forsberg and
Kjellén, 2001
).
Different roles for HS or heparin in Ca2+ kinetics have been
suggested, such as the buffering of Ca2+
(Bezprozvanny et al., 1993),
influencing growth factor signaling (Patel
et al., 1998
), or regulating the activity of ion channels such as
the dihydropyridine receptor (DHPR) (Knaus
et al., 1990
; Lacinova et al.,
1993
; Martinez et al.,
1996
) and the ryanodine receptor
(Bezprozvanny et al., 1993
;
Ritov et al., 1985
). The
availability of mice deficient in NDST-1 and NDST-2 provides a means to
further investigate the role of HS in skeletal muscle Ca2+ kinetics
in a physiological setting.
In the present study, we report on the effects of the HS deficiency on electrically induced Ca2+ spikes. Immunostaining for specific HS epitopes is reduced in skeletal muscle and in cultured myotubes of NDST-1/, but not in other genotypes. NDST-1/ myotubes show a significant decrease in the amplitude of Ca2+ spikes and a slower removal of cytosolic Ca2+. These results strongly indicate the involvement of HS in skeletal muscle Ca2+ kinetics.
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Materials and Methods |
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Anti-heparan sulfate antibodies
Preparation of phage display-derived c-Myc-tagged anti-HS antibodies
AO4B05, AO4B08, AO4F12, RB4CB9, RB4CD12, RB4EA12, and MPB01 was performed as
previously described (Jenniskens et al.,
2000). Control antibody MPB01 was randomly picked from phage
display `scFv library #1' (Nissim et al.,
1994
). This antibody does not recognize HS, or heparin, and is not
reactive with any of the cells or tissues tested (muscle, brain, kidney, lung)
as judged by ELISA and immunohistochemistry.
Mouse skeletal muscle tissue
Mice heterozygous for NDST-1 and NDST-2 were intercrossed to generate
offspring of all genotypes as previously described
(Forsberg et al., 1999;
Ringvall et al., 2000
). Both
strains have a mixed C57BL/6 and 129/SvJ/Sv genetic background. The
NDST-1/ mice were obtained from the F4-8 generation,
following back-crossing against C57BL/6, while the NDST-2+/
mice were all of the F6 generation. The first day of occurrence of a vaginal
plug was taken as day 0 of gestation (E0). E18.5 (18.5 days in utero) embryos
were isolated by caesarian delivery and killed by decapitation, in accordance
with the ethic regulations of the University. Embryos were rinsed in PBS,
snap-frozen in liquid nitrogen-cooled isopentane, and stored at
80°C.
Immunohistochemistry
Cryosections of whole embryos were cut (5 to 10 µm thick), mounted on
slides, dried thoroughly, and stored at 80°C until use. Cell
cultures were washed three times with PBS, dried overnight, and stored at
80°C until use. Cryosections and cell cultures were stained with
anti-heparan sulfate, anti-perlecan, and anti-DHPR antibodies, and with
-bungarotoxin as previously described
(Jenniskens et al., 2000
).
Photographs were taken, using a constant shutter time, on a Zeiss Axioskop
immunofluorescence microscope (Göttingen, Germany). For each genotype,
skeletal muscle specimens of individual embryos of two separate litters were
studied.
Structural analysis of HS from wild-type and NDST-1-deficient muscle
tissue
Embryos from a crossing of heterozygous mice (NDST-1+/)
were killed and genotyped at embryonic day 18.5. Hind leg muscle tissue was
isolated and the samples were lyophilized. The average dry weight was 5.7 mg.
HS from the samples was isolated and degraded by enzymatic cleavage, and the
disaccharides obtained were separated by reverse phase ion-pair
chromotography, followed by labelling with 2-cyanoacetamide, as previously
described (Staatz et al.,
2001). The obtained disaccharides were separated by reverse phase
ion-pair chromatography, followed by labeling with 2-cyanoacetamide. The
disaccharides were detected fluorometrically.
Primary myoblast culture
To obtain primary myoblast cultures, E18.5 embryos were isolated by
caesarian delivery, killed by decapitation, and dissected (disposed of skin
and other organs). Skeletal muscle tissue was isolated from the remaining
carcasses, triturated with needles and incubated in an enzyme solution (300 U
collagenase/ml, 0.15% trypsin, and 0.08% BSA in PBS, pH 7.3) at 37°C for
15 minutes. To the cell suspension, containing satellite cells (precursors of
myoblasts), an equal volume of neutralization solution (DMEM/50% fetal bovine
serum [FBS]) was added. The dissociation procedure was repeated three times.
Subsequently, cell suspensions were sieved through a 40 µm cell strainer
and cells were collected by centrifugation (10 minutes, 50 g).
Cell pellets were resuspended and enriched for myoblasts by pre-plating twice
for 30 minutes, after which cells were seeded at 5x104 cells
per well in a six-well format. Primary myoblast cultures were maintained as
proliferating cells for up to six passages in DMEM supplemented with 10% brain
extract and 1% Ultroser G (Portiér
et al., 1999). Cells were plated at 2x105
(six-well format) or 5x104 (24-well format) cells per well
and grown overnight in proliferation medium to reach 50-60% confluency the
next day. At confluency, culture medium was replaced by differentiation medium
(DMEM/10% brain extract/0.4% Ultroser G). Differentiation medium was refreshed
every second day. For immunocytochemistry, H2SO4-etched
13 mm diameter coverslips were used in 24-well format. For measurements of
cytosolic Ca2+, cells were plated on
H2SO4-etched 24 mm diameter glass coverslips in six-well
format. Cultures were grown and differentiated at 37°C in a humidified 5%
CO2, 95% air atmosphere. For each genotype, primary myoblast
cultures derived from at least two individual embryos of two (NDST-2) or three
(wildtype and NDST-1) separate litters were studied.
Ratiometric measurements of intracellular Ca2+ with
Indo-1
Coverslips with confluent monolayers of primary myoblast cultures,
differentiated for three days, were washed twice with physiological salt
solution (PSS; 125 mM NaCl, 10 mM NaHCO3, 1 mM
NaH2PO4, 5 mM KCl, 2 mM MgSO4, 1.8 mM
CaCl2, 10 mM HEPES and 10 mM glucose, pH 7.4) and loaded with
Indo-1 using an Indo-1/AM solution (5 µM Indo-1/AM and 1 µM Pluronic
F127 in PSS) for 45 minutes. Cell loading was performed at 37°C in a
humidified 5% CO2, 95% air atmosphere. Cells were washed twice with
PSS, placed in a Leiden chamber (Ince et
al., 1985), and incubated in PSS for another 15 minutes. The
Leiden chamber was placed on a Nikon diaphot inverted microscope attached to
an OZ confocal laser scanning microscope (Noran instruments, Madison, WI).
Electrical stimulation of myotubes was performed at 15 to 20 V with 10
milliseconds duration and 0.025 milliseconds delay. Repetitive stimuli were
generated at a frequency of 1 Hz. Stimulation was performed using a pair of
parallel oriented platinum electrodes (5 mm interspaced, placed beside the
monolayer), connected to a Grass SD9 electric pulse generator. Ca2+
free medium was obtained by the use of Ca2+-free PSS, supplemented
with 1 mM EGTA. For ratiometric Ca2+ recordings, Indo-1 was excited
at 351 nm using a hardware-modified high power argon-ion laser (Coherent
Enterprise, Santa Clara, CA). After being separated by a 455 DCLP dichroic
mirror, Indo-1 fluorescence intensity was collected at 405±45 nm and
485±45 nm using photo multipliers. All measurements were performed at
room temperature, using a Nikon x40 water-immersion objective, NA 1.2
with high UV transmission. The estimated laser intensity at the end of the
objective lens was 28 µW. Confocality (optical section thickness: 1 to 2
µm) was achieved by using a slit width of 100 µm
(Koopman et al., 2001
).
User-based hardware set-up and data acquisition was controlled by Intervision
software (Version 1.5, Noran Instruments), running under IRIX 6.2 on an Indy
workstation (Silicon Graphics, Mountain View, CA) equipped with 128 MB of RAM.
To improve signal-to-noise ratio, single full-frame images (512x480
pixels) were collected at 30 Hz by averaging of 32 images for each Indo-1
emission wavelength. Pairs of fluorescence intensities were saved in SGI-movie
(mv) format, ready for off-line analysis using the Intervision 2D software
package (Noran Instruments). Regions of interest (ROI) were drawn and
numerical values were stored in ASCII-format. Additional data-analysis was
carried out by using Origin 6.0 (Microcal, Northampton, MA), supplemental
image analysis and visualization were performed with Image Pro-plus 4.1 (Media
Cybernetics, Silver Spring, MD).
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Results |
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|
|
When compared to other genotypes, the diameter of myofibers was slightly less uniform in NDST-1/ muscle, whereas the overall muscle fiber morphology seemed normal. The occurrence and size of neuromuscular junctions (NMJs) were normal, as judged from the staining of acetylcholine receptor clusters. Similarly, staining intensities remained virtually unaltered for other ion channels involved in skeletal muscle excitation-contraction coupling, e.g. the voltage gated sodium channel (data not shown), the DHPR (Fig. 2c,d), and the ryanodine receptor (data not shown). The occurrence and the distribution of the HSPG perlecan core protein was not affected in NDST-1/ muscle (Fig. 2e,f).
Disaccharide composition of HS from wild-type and NDST-1-deficient
muscle tissue
HPLC analysis of fully digested HS, isolated from wild-type and
NDST-1/ skeletal muscle, indicated a decrease in
sulfation in NDST-1/ muscle
(Fig. 3). The amount of
N-acetylated HS disaccharides was significantly increased and the
level of N-sulfation was significantly reduced. Also,
2-O-sulfation was lower, whereas 6-O-sulfation was normal.
These results are similar to those obtained in studies of HS from wild-type
and NDST-1-deficient liver (J.L., M.R., L.K. et al., unpublished).
|
Culturing of primary myoblasts
Primary myoblast cultures were generated from muscle tissue of E18.5
embryos. At three days of differentiation, multinucleated myotubes were
present in all cultures. From day three of differentiation onwards,
spontaneous contractions were regularly observed in wild-type and heterozygous
myotubes, but only occasionally in knockout myotubes. In wild-type and
heterozygous cultures, spontaneous contracting myotubes caused monolayers to
peel off from the culture plastics, an effect that was not observed in
NDST-1/ and NDST-2/
cultures.
Occurrence of HS epitopes in vitro
To analyze the occurrence of HS epitopes in cultured myotubes,
three-day-differentiated cultures were stained immunologically. All anti-HS
antibodies stained the BL of wild type
(Fig. 4a,e) and heterozygous
(data not shown) myotubes. Normal BL staining was present in
NDST-2/ cultures
(Fig. 4g). However,
NDST-1/ cultures showed hardly any HS staining, even
at sites of spontaneous acetylcholine receptor clustering
(Fig. 4c), indicating the
inability of NDST-1-deficient myotubes to synthesize the HS epitopes
recognized by the antibodies.
|
The clustering of acetylcholine receptors appeared normal in muscle
cultures of all genotypes, as judged by -bungarotoxin staining
(Fig. 4, arrows). As was also
shown in situ, the in vitro occurrence of the HSPG perlecan core protein was
not affected by NDST-1 or NDST-2 deficiency (data not shown).
Electrically induced Ca2+ spikes in NDST-1/-2 affected
myotubes
Ratiometric measurements of the cytosolic Ca2+ concentration
([Ca2+]i) were performed in myotubes of all genotypes,
using a high-speed UV confocal laser scanning microscope and electrical
stimulation. Average amplitudes of electrically induced Ca2+ spikes
decreased gradually in the following order: wild type >
NDST-2+/ > NDST-2/ >
NDST-1+/ > NDST-1/, but only
NDST-1/ differed significantly form the wild type
(Table 1,
Fig. 5). All Ca2+
spikes were independent of the presence of Ca2+ in the
extracellular environment, indicating that the released Ca2+
originated from intracellular stores. Upon electrical stimulation,
[Ca2+]i reached its maximum within 99 milliseconds
(three data points), after which it rapidly declined to a basal level
(Fig. 5), with mono-exponential
kinetics (Yt=Y0+Aet/µ
where Yt= ratio as a function of time {reflecting the
[Ca2+]i}; Y0= ratio at t=0 {basal ratio,
reflecting the basal [Ca2+]i}; A= scaling factor; t=
time; µ= decay constant) using the Levenberg-Marquard algorithm
(Press et al., 1992). This
mono-exponential description was valid because of the small difference between
the experimental trace and the fitted exponential function
(R2=0.98) and the small s.e.m. of the average µ
(0.26±0.03). The mono-exponential kinetics suggest one main
[Ca2+]i removal process
(Koopman et al., 2001
;
Lieste et al., 1998
;
de Groof et al., 2002
),
described by the decay constant µ, which is inversely proportional to the
rate of [Ca2+]i decline
(Table 1).
NDST-1/ myotubes differed significantly from
wild-type myotubes in decay constant (µ; P<0.005). Together
with the reduced Rmax this indicates that in these myotubes the quantity of
released Ca2+ and the [Ca2+]i removal rate
are affected (Table 1;
Fig. 5). By plotting µ as a
function of Rmax, it was found that the Ca2+ removal rate (i.e.
1/µ) was positively correlated to Rmax. Only in
NDST-1/ myotubes Ca2+ spikes were
occasionally skipped upon electrical stimulation, especially when stimulated
at high frequency (>1 Hz).
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Discussion |
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Although staining for HS epitopes was considerably reduced in skeletal muscle from NDST-1/ mice, the HS content of neural BL and the synaptic BL at the NMJ appeared unaffected, indicating a different HS profile of the latter BLs. Other NDST isoforms may be active in the highly specialized NMJ and neural areas. No obvious effects on the presence of HS epitopes were seen in mice heterozygous for NDST-1, indicating a compensatory effect of the single functional allele present. Also, no effect was observed in NDST-2/ mice or in mice heterozygous for NDST-2. Overall morphology of NDST-1/ muscle appeared to be normal, based on immunohistological staining for the ion channels of the excitation-contraction coupling cascade (acetylcholine receptor clusters, voltage gated sodium channels, DHPR, and ryanodine receptor) and for HSPG core protein perlecan, which is present in the BL.
In primary myoblast cultures, staining for HS epitopes in NDST-1/ cultures was virtually absent, whereas in NDST-2/ cultures, or in cultures derived from heterozygous mice, staining was normal. The occurrence and size of acetylcholine receptor clusters on myotubes appeared similar in all genotypes, indicating that the loss of either enzyme does not interfere with the proper clustering of this receptor. Also, perlecan core proteins were distributed normally, arguing for a normal protein constitution of the extracellular environment of cultured myotubes.
Ca2+ kinetics was affected to various degrees in myotubes of
individual cultures. The rate of [Ca2+]i removal
correlated with the amplitudes of Ca2+ spikes, both of which were
significantly decreased for NDST-1/ myotubes. The
lower amplitude in NDST-1/ myotubes is indicative of
a lowered Ca2+ flux over the sarcoplasmic reticulum membrane.
Heparin, a highly sulfated HS, binds with high affinity to the extracellular
domain of the voltage-dependent L-type Ca2+ channel (the DHPR), of
skeletal muscle cells (Knaus et al.,
1990). Upon stimulation, this receptor induces a conformational
change in the Ca2+ release channel of the sarcoplasmic reticulum
(the ryanodine receptor), resulting in a Ca2+ flow from the
sarcoplasmic reticulum to the cytosol. HS might be involved in the correct
functioning of the dihydropyridine receptor and alterations in HS might reduce
the Ca2+ flow to the cytosol. Alternatively, the effect of HS could
be indirect, e.g. by modulating growth factor signaling. The effect of
decorin, a dermatan sulfate proteoglycan, on Ca2+ kinetics is
mediated through the EGF receptor (Patel
et al., 1998
). Also, there might be a lowered Ca2+
concentration in the sarcoplasmic reticulum (and thus a lowered
Ca2+ flux) as a result of the NDST-1/
deficiency. Also, the [Ca2+]i removal rate is affected
in NDST-1/ myotubes. Since this process is mainly
effectuated by sarcoplasmic reticulum Ca2+ ATPases, an effect of HS
deficiency on the overall Ca2+-pumping rate of this ion pump could
be envisioned. The occurrence of skeletal muscle differentiation
characteristics such as fusion to multinucleated myotubes, spontaneous
acetylcholine receptor clustering, and excitability indicate that myotubes of
all genotypes were differentiated to a similarly degree. Therefore, the
effects observed are not likely the result of disturbed differentiation.
NDST-1-deficient cultures occasionally failed to generate Ca2+
spikes upon electrical stimulation. Such aberrant Ca2+ kinetics
were not observed in any other genotype, arguing for these effects to be a
result of the loss of this enzyme. NDST-1/ mice die
shortly after birth due to respiratory failure. This effect has been
attributed to immature type II pneumocytes, resulting in shortage of lung
surfactant (Fan et al., 2000;
Ringvall et al., 2000
).
However, the loss of muscle contraction, likely to be the result of disturbed
Ca2+ kinetics, may also contribute to the lack of pulmonary
function.
In this study, we report the effects of the deficiency of NDST-1 or NDST-2 on electrically induced Ca2+ spiking in skeletal muscle. The results presented here strongly indicate the involvement of HS in skeletal muscle Ca2+ kinetics.
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Acknowledgments |
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