Department of Biology and Center for Molecular Genetics, University of California San Diego, La Jolla, California 92093-0357
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Abstract |
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Spontaneous calcium release from intracellular stores occurs during myofibrillogenesis, the process of sarcomeric protein assembly in striated muscle. Preventing these Ca2+ transients disrupts sarcomere formation, but the signal transduction cascade has not been identified. Here we report that specific blockade of Ca2+ release from the ryanodine receptor (RyR) activated Ca2+ store blocks transients and disrupts myosin thick filament (A band) assembly. Inhibition of an embryonic Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) by blocking the ATP-binding site, by allosteric phosphorylation, or by intracellular delivery of a pseudosubstrate peptide, also disrupts sarcomeric organization. The results indicate that both RyRs and MLCK, which have well-described calcium signaling roles in mature muscle contraction, have essential developmental roles during construction of the contractile apparatus.
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Introduction |
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TRANSIENT elevations of intracellular calcium (Ca2+)
are an information transfer mechanism (Berridge,
1997; Spitzer and Sejnowski, 1997
) that may be conserved during cellular differentiation, because they can
regulate cytoskeletal organization (Kater et al., 1988
; Rees
et al., 1989
; Gomez et al., 1995
) and gene expression
(Sheng et al., 1990
; Dolmetsch et al., 1997
; Fields et al.,
1997
). For example, spontaneous Ca2+ elevations observed
in Xenopus spinal neurons are necessary for normal differentiation in culture, since blocking these transients prevents normal extension of neurites, maturation of potassium current kinetics, and development of GABA immunoreactivity (Gu et al., 1994
; Gu and Spitzer, 1997
). Moreover,
imposed Ca2+ transients are necessary and sufficient to
promote these aspects of neuronal differentiation in a frequency-dependent manner (Gu and Spitzer, 1995
).
Many developmental studies have focused on the role of
Ca2+ signaling in early eventswaves after fertilization
(Busa and Nuccitelli, 1985
; Galione et al., 1993
; Gillot and
Whitaker, 1993
; Jaffe, 1995
) or Ca2+ transients in blastomeres during cytokinesis (Reinhard et al., 1995
; Muto et al.,
1996
; Silver, 1996
; Webb et al., 1997
). If Ca2+ transients
are a signaling mechanism used throughout development, then many tissues undergoing primary differentiation
should exhibit them, and distinct patterns of transients
could be correlated with cell type. In support of this view,
spontaneous Ca2+ transients occur in embryonic Xenopus
myocytes both in culture and in vivo and have been shown
to regulate myofibrillogenesis (Ferrari et al., 1996
; Ferrari,
M.B., and N.C. Spitzer. 1997. Dev. Biol. Abstr. 186:337a).
Skeletal muscle is an excellent system for studying general
mechanisms of Ca2+-dependent cytoskeletal processes;
contractile and associated proteins are assembled in highly
ordered units called sarcomeres during the process of
myofibrillogenesis so that disruptions of this lattice are
readily apparent.
Cell-free systems have been used to examine myofilament and myofibril dynamics and protein turnover
(Bouche et al., 1988), demonstrating that some sarcomeric
proteins are in dynamic equilibrium with the cytosol.
However, myofibril construction in a cell-free system has
not yet been achieved. This may indicate that formation of
this complex apparatus does not obey simple rules of self-assembly used for first order processes such as G- to F-actin
assembly or polymerization of tubulin to form microtubules. Our previous work supports the hypothesis that second messenger systems are involved in coordinating the
spatial arrangement of contractile proteins and their subsequent organization, since the formation of myofibrils is
significantly disrupted when Ca2+ transients are blocked
(Ferrari et al., 1996
).
How myocyte Ca2+ transients are generated and what
downstream mechanisms facilitate formation of sarcomeres are currently unknown. At least a dozen Ca2+-dependent proteins may be involved in myofibrillogenesis (Epstein and Fischman, 1991), thus providing a framework for
Ca2+-mediated sarcomere assembly. We focused on the
mechanisms of Ca2+ release and the potential role of
Ca2+-dependent kinases, and report here that ryanodine
receptors (RyRs)1 and myosin light chain kinase (MLCK),
in addition to their well-documented roles in the Ca2+-dependent contraction of adult muscle, play novel developmental roles during myofibrillogenesis.
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Materials and Methods |
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Myocyte Cultures
Myocytes were cultured from neural plate (stage 15) Xenopus embryos;
this stage is several hours after cells have completed their final cell cycle
and acquired the ability to differentiate autonomously (Kato and Gurdon,
1993). Embryos were split mid-sagittally to be plated in paired dishes for
experimental versus control conditions using established techniques
(Spitzer and Lamborghini, 1976
; Kidokoro et al., 1980
; Ferrari et al.,
1996
). The posterior neural plate with adjacent lateral regions was excised
and placed in a divalent cation-free medium ([mM] 117 NaCl, 0.7 KCl, 4.6 Tris, 0.4 EDTA, pH 7.8) for 20-30 min to promote disaggregation. Cells
were gently aspirated and plated in 35-mm tissue culture dishes (Costar
Corp., Cambridge, MA) in standard (control) saline ([mM] 117 NaCl, 0.7 KCl, 1.3 MgCl2, 2 CaCl2, 4.6 Tris, pH 7.8) or zero-calcium [0-Ca2+] saline
(as above, no added CaCl2 with 2 mM EGTA). These mixed cultures contain myocytes, neurons, and morphologically undifferentiated cells.
Unless otherwise noted, all pharmacologicals were applied from 6 to 24 h
in culture, 3-6 h before A band assembly begins (see Fig. 2 A), or 24-48 h
in culture, when myocytes have functionally differentiated. Treatments
for shorter periods were followed by 10 washes of 4 ml each (culture bath
volume was 2 ml). Kinase inhibitors were used at concentrations only
2-10-fold above their reported inhibition constant (Ki) values to enhance the
likelihood of specificity for individual kinases. Only bipolar myocytes, with length 4× width, were examined; multipolar cells and myoballs were
not considered. Myocytes were screened for apoptosis using a DNA fragmentation detection system (ApopTag Plus; Oncor, Gaithersburg, MD).
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Calcium Imaging
Measures of resting Ca2+ and spontaneous transients were achieved with
fura-2 and fluo-3, respectively, using established techniques (Tsien, 1980;
Grynkiewicz et al., 1985
; Gu et al., 1994
; Ferrari et al., 1996
). Cells were
loaded with calcium indicator by bath application (2 µM, solubilized in
<0.1% DMSO). Indicator loading time was 30-60 min, cultures were then
washed in 5-10 volumes of standard saline, and images were collected at
10-s intervals. Zeiss Neofluor water immersion objectives (Carl Zeiss Inc.,
Thornwood, NY) were used for both calcium imaging and indirect immunofluorescence. Ratiometric measures of resting Ca2+ were made using
fura-2 visualized with a Zeiss Photomicroscope and Dage 72SX ICCD
camera. Cells were imaged between 3 and 9 h after plating in control, 100 µM
ryanodine, and 0-Ca2+ cultures. Myocytes were imaged at 10-s intervals
for 10 min, and these numbers were averaged for baseline values; details
of data acquisition and analysis were previously described (Ferrari et al.,
1996
). Non-ratiometric measures of fluo-3 signals and immunofluorescence were made with an MRC 600 confocal laser system (Bio-Rad Laboratories, Hercules, CA). Images were digitized and saved using the COMOS
program (Bio-Rad Laboratories), and analyzed using macros for the National Institutes of Health (NIH) Image program (version 1.47; W. Rasband, NIH, Bethesda, MD) as previously described (Ferrari et al., 1996
).
Myocytes were imaged for spontaneous transients 1-2 h after incubation
in the same concentration of kinase inhibitor that disrupted myofibrillogenesis.
Immunochemistry
Sarcomeric myosin was visualized with the mAb MF20 (Developmental
Studies Hybridoma Bank, the University of Iowa, Iowa City, IA) and an
FITC-conjugated secondary antibody. For immunoblots, antibodies to
smooth MLCK isoforms (R57, K36; gifts of P. Gallagher, Indiana University School of Medicine, Indianapolis, IN) were used because they recognize a combination of smooth 130-kD, embryonic 208-kD, and 220-kD
MLCK isoforms in avian and mammalian tissues and cell lines (Gallagher
et al., 1995). MLCK isoforms were examined from homogenates of Xenopus embryonic and adult tissues; each lane was loaded with 100 µg of protein. Dilutions of the primary and secondary antibodies were determined
empirically, and labeled bands were detected using the enhanced chemiluminescence detection system (Amersham Corp., Arlington Heights, IL).
Sarcomere Assays
Sarcomere (A band) numbers were counted at 400× in 45 myocytes in
blind assays from
3 culture pairs for each condition at 24 or 48 h. Digital
images of sarcomeric myosin immunofluorescence were captured on the
Bio-Rad MRC 600 using a fluorescein filter set. Thin optical sections
(minimum aperture; 12-16 sections per cell) were taken with a focus motor in 1.2-µm steps. Numbers of bipolar cells were counted in six to nine
dishes. In all cases n is the same for control and experimental data. Raw
data from paired controls were used for statistical tests.
Electrophysiology
Myocyte inward rectifier potassium current was measured in 12 cells
grown for 24 h in culture under various conditions, using standard techniques (Spruce and Moody, 1992
; Ferrari et al., 1996
). Pipettes contained
(mM): 100 KCl, 10 NaCl, 5 EGTA, 10 Hepes, 2 MgATP, 20 KOH, pH 7.4, and had resistances of 2-4 M
. External recording saline contained
(mM): 117 NaCl, 3 KCl, 2 CaCl2, 5 Hepes, 2 NaOH, pH 7.4. Potassium
currents were isolated using 0.2 µg/ml tetrodotoxin and 0.4 mM CdCl2 in
the external solution to block Na+ and Ca2+ currents, respectively. Current was recorded with a series of 30-ms voltage steps (
130 to +40 mV)
from a holding potential of
50 mV. Leak-subtracted current traces were
filtered at 3-5 KHz and digitized at 20 KHz. Steady-state mean current
amplitudes were measured over the last 5 ms of the voltage step. Currents are expressed as pA/pF to normalize for cell size.
Synthetic Peptides
The MLCK pseudosubstrate inhibitory amino acid sequence (MLCKi),
AKKLSKDRMKKYMARRKWQKTG, is highly conserved, being identical at the amino acid level among the known vertebrate smooth MLCK isoforms (BlastP Search of GenBank). This inhibitory sequence has Ki
values of 0.3-21 nM in vitro (Ikebe et al., 1992). A scrambled version of
this peptide, KKDTQWMYLKMRKGRAKSAKRK, showed no significant homology to any GenBank sequences. MLCKi and the scrambled
version were each fused to an internalizable peptide of the homeodomain antennapedia protein (pANT 43-58: RQIKIWFQNRRMKWKK) by a
disulfide linkage (Theodore et al., 1995
; Prochiantz, 1996
). Peptides were synthesized, conjugated, and then analyzed at the Stanford Beckman Center's Protein & Nucleic Acid facility. Antennapedia peptide or the conjugates were applied at 100 nM for 1 h at 6 or 24 h in culture.
Statistics
Measurements are reported as mean ± SEM. Unpaired two-tailed t tests were used and values are considered significantly different for P < 0.05.
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Results |
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Ryanodine Receptor Stores Are Necessary for Transients and Myofibrillogenesis
Transients are produced from intracellular Ca2+ stores
and are required for normal sarcomere assembly (Ferrari
et al., 1996), suggesting Ca2+ influx may not be necessary
for myofibrillogenesis. In support of this hypothesis, myocytes grown in 0-Ca2+ for 24 or 48 h in culture have normal
bipolar morphology as well as myofibrillar and sarcomeric
structure (Fig. 1 A). This arrangement is clearest in the
myocyte endfeet, which are free of yolk inclusions. The
numbers of bipolar myocytes (0-Ca2+ 76 ± 11% of controls, P = 0.24) and the numbers of sarcomeres per myocyte in 0-Ca2+ cultures (0-Ca2+ 96 ± 8% of controls, P = 0.68; Figs. 1 A, and 2 B) do not differ from controls grown
in 2 mM Ca2+. Since Ca2+ transients are blocked by ryanodine (Ferrari et al., 1996
; Fig. 1 B), we tested whether
blocking the RyR Ca2+ store results in myofibrillar defects. Ryanodine (100 µM) in standard 2 mM Ca2+ saline,
applied from 6 to 24 or 6 to 48 h in culture, reduces the
number of sarcomeres per cell (6-24 h ryanodine 66 ± 6%
of controls, P = 0.006; Figs. 1 B, and 2 B) and disrupts myofibril alignment; most myofibrils do not span the length of
the cell and are not in close parallel register.
|
Intracellular Ca2+ was monitored in response to ryanodine
treatment. Ryanodine blocks transients without affecting
baseline Ca2+; fura-2 measures in 100 µM ryanodine-treated myocytes are not significantly different from controls ([nM] ryanodine 62 ± 9, control 42 ± 4, P = 0.06; Fig.
1 C), in agreement with previous control values (24-64
nM; Ferrari et al., 1996). In addition to these steady-state
values, measurements of Ca2+ during acute ryanodine application show that resting levels are not altered, but a single spontaneous transient can still occur before complete
block (data not shown). A use-dependent block is expected for this agent, as high concentrations of ryanodine antagonize by binding a low affinity site only when the receptor is in the open configuration (Meissner, 1986
).
Ryanodine is a highly specific toxin (Coronado et al.,
1994), and does not affect the total number of bipolar
myocytes per dish (6-24 h ryanodine 122 ± 16% of controls, P = 0.35; 6-48 h ryanodine 103 ± 24% of controls, P = 0.94). Moreover, ryanodine has no effect on the normal
maturation of inward rectifier potassium current during
the period of transient production (Fig. 1 D). We chose
the inward rectifier as a positive control because it is the
earliest developing voltage-gated current in Xenopus myocytes, with an increase in current density occurring in parallel with the period of myofibrillogenesis (Spruce and
Moody, 1992
).
The period of transient production precedes A band formation (Fig. 2 A). Brief ryanodine treatment during this time, from 3 to 6 h, or 3 to 15 h in culture, also disrupts thick filament assembly (Fig. 2 B). These results indicate that later synthesis and insertion of new RyRs does not compensate for blocking calcium release during this sensitive period. Since myofibrillogenesis is perturbed by ryanodine during transient production and initial assembly, but not at later times (Fig. 2 B), ryanodine does not exert its effects by promoting disassembly.
Identification of a Downstream Kinase Necessary for Myofibrillogenesis
Do Ca2+ transients regulate myofibrillogenesis via activation of kinases? Staurosporine (100 nM), a general serine/
threonine kinase inhibitor, reduces numbers of A bands
(56 ± 6% of controls, P = 0.001; Fig. 3, A and C) and results in a diffuse distribution of sarcomeric myosin in the
cell soma. Thin processes and lamellipodial-like structures
emanate from the myocyte endfeet with dense punctate myosin at the edge of these membranes. This result encouraged use of more specific blockers that inhibit Ca2+-dependent kinases. Convergent results with multiple inhibitors of MLCKML-7, ML-9, and KT5926
implicate
this Ca2+/calmodulin-dependent enzyme as an effector in
myofibrillogenesis (Fig. 3 C). These inhibitors also produce a diffuse distribution of myosin with some dense accumulations, and sarcomere numbers are significantly reduced (ML-7 [1 µM] 47 ± 15% of controls, P = 0.0001;
Figs. 3, B and C). Inhibitors of protein kinase A, protein kinase C (PKC), and Ca2+/calmodulin kinase II (CaMK II)
are without effect (Fig. 3 C). ML-7, the most specific of the
MLCK inhibitors tested, disrupts myofibrillogenesis without affecting development of the inward rectifier potassium current (Fig. 3 D). ML-7, ML-9, KT5926, and staurosporine also do not affect normal generation of Ca2+
transients when examined between 3 and 9 h in culture
(data not shown), nor do these inhibitors affect the number of bipolar myocytes differentiating per culture (e.g.,
ML-7 [1 µM] 78 ± 16% of controls, P = 0.39).
|
Myocytes treated with ryanodine or kinase inhibitors appear normal under phase optics, having phase dark endfeet, birefringent yolk inclusions, clear nuclei, and no membrane blebbing. Since cytoskeletal disruptions could result from pharmacologically triggered apoptosis, myocytes treated with ryanodine or ML-7 from 6 to 48 h in culture were screened for DNA fragmentation, a late marker of apoptosis. Although some pyknotic cells and cellular fragments in these mixed cultures had labeled nuclei, neither the number of bipolar myocytes, nor the number of labeled nuclei in myocytes, differed from controls (<3% labeled nuclei in n > 100 myocytes for each condition).
MLCK Activity Is Suppressed by Activation of PKC
Activation of PKC inhibits MLCK activity in vitro by
phosphorylating its only known substrate, the regulatory
light chain (RLC), preventing MLCK access to the RLC
(Nishikawa et al., 1984; Turbedsky et al., 1997
). To test the
hypothesis that activation of PKC generates the phenotype produced by MLCK inhibitors, we stimulated PKC
with phorbol 12-myristate, 13-acetate (PMA). Application of 10 nM PMA from 6 to 24 h in culture disrupts myofibrillogenesis and reduces sarcomere numbers (PMA 30 ± 7%
of controls, P = 0.0001; Fig. 4). This disruption is not seen
with PMA application from 24 to 48 h in culture (Fig. 4 C).
Moreover, PMA-activated inhibition of sarcomere assembly is blocked by co-application of PMA with the PKC inhibitor bisindolylmaleimide I (Bis I; 100 nM) (PMA/Bis I
85 ± 10% of controls, P = 0.35; Figs. 4, B and C), rescuing normal differentiation.
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Molecular Inhibition of MLCK Activity In Situ by a Pseudosubstrate Peptide
Vertebrate MLCKs contain a conserved autoinhibitory
domain (Gallagher et al., 1997), and pseudosubstrate peptides from this region inhibit MLCK activity in biochemical assays (Ikebe et al., 1992
; Knighton et al., 1992
). We
treated myocytes with a 22-amino acid synthetic inhibitory peptide (MLCKi) from this region conjugated to a
16-amino acid homeopeptide of the antennapedia gene
(pANT) to transport it into cells (Theodore et al., 1995
;
Prochiantz, 1996
). As with pharmacological inhibitors,
myofibril arrays are disrupted and sarcomere numbers are
reduced with pANT-MLCKi ([100 nM] 34 ± 6% of controls, P = 0.0001; Figs. 5, A and C). Some normal A bands
are visible within disrupted myofibrils and regions of dense
myosin accumulation are evident. Treatments with pANT
alone, pANT coupled to a scrambled MLCKi sequence, or
later application of pANT-MLCKi have no significant effect on myofibril alignment or sarcomere numbers, showing specificity both with respect to the inhibitory sequence
of amino acids and the time of application (Fig. 5 C).
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Detection and Developmental Regulation of MLCK Isoforms
Which MLCK is present in myocytes during the period of
A band assembly? An embryonic MLCK is expressed in
developing skeletal muscle and other tissues, and R57
polyclonal antiserum recognizes both smooth and embryonic isoforms (Gallagher et al., 1995). This antiserum identifies a single band at 225 kD on immunoblots of Xenopus
myotomal tissue during the period of sarcomere assembly (Fig. 6 A). This Xenopus MLCK isoform shows developmental upregulation during the period of myofibrillogenesis corresponding to production of transients, but is absent
in adult skeletal tissue. These blots also show the presence
of a 130-kD Xenopus smooth MLCK in adult skeletal, but
not embryonic, tissue. The mAb K36 also recognizes these
130- and 225-kD isoforms, giving the same pattern on immunoblots seen with R57 (data not shown). Since only one
embryonic MLCK isoform is recognized by these antibodies, we determined its cellular distribution in cultured myocytes using the K36 mAb. While immunofluorescence was
weak compared with myosin immunoreactivity with MF20,
either because of lower levels of the epitope and/or lower
affinity of the primary antibody, the 225-kD embryonic
MLCK is localized in a striated pattern (Fig. 6 B).
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Our findings are summarized by a signal transduction cascade schematic that includes the components we have identified (Fig. 6 C). Intervention at the different points indicated suppresses the assembly of sarcomere A bands. These results are expected to be useful in decoding the algorithm by which Ca2+ transients enable cytoskeletal organization.
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Discussion |
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Our previous work led to the hypothesis that Ca2+ transients direct some aspects of myofibrillogenesis in embryonic Xenopus myocytes (Ferrari et al., 1996). Since Ca2+
transient production does not depend on extracellular
Ca2+, a corollary is that removal of extracellular Ca2+
should not disrupt construction of the contractile apparatus. We show here that myocytes grown for
48 h in the
absence of Ca2+ have no defects in myofibril orientation
and sarcomere assembly. This suggests that voltage-dependent Ca2+ channels and other Ca2+ influx pathways do not
play a role in the early stages of myofibrillogenesis, although they may participate later in the accelerated formation of striations observed in twitching cells (Kidokoro
and Saito, 1988
).
Ryanodine disrupts myofibril alignment and reduces
sarcomere numbers in a manner both qualitatively and
quantitatively similar to that achieved by BAPTA-AM
blockade of transients (Ferrari et al., 1996). Since ryanodine blocks transients without affecting the total number
of bipolar myocytes per culture, resting Ca2+ levels, or the
development of the potassium inward rectifier current, it is
a highly specific tool for examining the role of transients in
muscle development. Structural defects similar to those reported here have been observed in two vertebrate RyR
mutants and in wild-type chicken muscle cultured in the
presence of chronic ryanodine (Airey et al., 1993a
,b;
Takekura et al., 1995
), suggesting that RyRs may have developmental roles (Airey et al., 1991
). In this regard, it is
notable that the sarcoplasmic reticulum develops in close
physical association with the formation of myofibrils (Huang and Hockaday, 1988
; Flucher et al., 1993
; Takekura
et al., 1994
). While not excluding the possibility that RyRs
play a structural role during development, our results indicate that RyRs play a physiological role, suggesting that
release of Ca2+ from this store is not only important for E-C
coupling in mature muscle, but also for proper construction of the contractile apparatus.
We initially used a pharmacological approach to determine if Ca2+-dependent kinases play a role in myofibrillogenesis. The results of this approach indicated that
MLCK, a Ca2+/calmodulin-dependent enzyme, is involved
in myofibrillogenesis. Inhibiting this kinase resulted in a
more severe phenotype than that produced by ryanodine.
Since ryanodine does not alter the resting Ca2+ concentration, one possibility is that basal MLCK activity persists in
ryanodine-treated cells. This is not unlikely, given the activation constant for MLCK by Ca2+/calmodulin is 1 nM
(Gallagher et al., 1997). This residual activity would be
blocked by MLCK inhibitors.
Further support for MLCK involvement was achieved
by activation of PKC, which indirectly inhibits MLCK.
The RLC is the only known substrate for MLCK, and
when phosphorylated by PKC, access to serine 19 of the
RLC by MLCK is blocked (Nishikawa et al., 1984; Turbedsky et al., 1997
). PKC activators were thus predicted to
disrupt myofibrillogenesis, as observed with PMA. This
result also indicates that the absence of an effect with the
PKC inhibitor Bis I was not due to the lack of PKC in
these cells. In fact, PKC-mediated inhibition of sarcomere
assembly was blocked by co-application of PMA with Bis I,
indicating that Bis I is an effective inhibitor of PKC in
these cells. These observations support the idea that PKC
activity acts as a negative regulator of MLCK activity, and implies that basal PKC activity remains low during striated
muscle development to allow for MLCK-mediated sarcomere (A band) assembly.
Despite the strength of the pharmacological results, we
sought more specific means of blocking MLCK activity to
verify its role in myofibrillogenesis. We used a synthetic
peptide from the conserved autoinhibitory domain of
smooth MLCK (Ikebe et al., 1992; Knighton et al., 1992
),
using an intracellular delivery method shown to be effective in blocking PKC activity in neurons (Theodore et al.,
1995
; Prochiantz, 1996
). Sarcomere assembly was affected
only by the inhibitory MLCKi peptide construct, whereas a scrambled version had no effect. This result indicates
that inhibition was due to MLCKi instead of a nonspecific
consequence of the antennapedia transport peptide or
cleavage by-products. Furthermore, later application of
MLCKi had no effect, consistent with the sensitive window already established with ryanodine and pharmacological inhibitors of MLCK.
The three major vertebrate MLCK isoforms that have
been described (smooth, skeletal, and embryonic) are all
Ca2+ dependent (for reviews see Trybus, 1994; Gallagher
et al., 1997
). Adult skeletal muscle expresses smooth muscle as well as skeletal muscle MLCK isoforms, whereas developing skeletal muscle expresses a newly discovered embryonic MLCK (Gallagher et al., 1995
). Our results suggest
amphibian skeletal muscle also expresses the embryonic MLCK isoform, since antibodies to smooth and embryonic
forms recognize only a single band of appropriate size in
embryonic tissue. In addition, the inverse developmental
regulation of the 130-kD Xenopus smooth muscle MLCK
and embryonic 225-kD isoforms is similar to that observed
in chicken and mammalian tissues (Gallagher et al., 1995
).
Structural motifs in smooth MLCK are similar to those
found in proteins of the N-CAM superfamily associated
with thick filaments, including the giant sarcomeric protein titin (Herring et al., 1990; Epstein and Fischman, 1991
;
Labeit and Kolmerer, 1995
), and smooth MLCK localizes
to myosin-containing structures (Guerriero et al., 1981
; de
Lanerolle et al., 1981
). If the embryonic MLCK contains
the same motifs, it is likely targeted to developing A
bands. The striated labeling produced by an MLCK antibody which recognizes a single embryonic isoform on immunoblots is consistent with this idea.
The myosin II isoform(s) that must be activated by
MLCK for A band assembly in skeletal muscle remain to
be determined. In cardiac myocytes, myosin IIB appears
in premyofibrils and is gradually replaced by sarcomeric
myosin (Rhee et al., 1994). Since MLCK activates nonmuscle myosin II and is responsible for assembly of "thick
filaments" in activated smooth muscle (Scholey et al., 1980
), this process may be a conserved early step in striated muscle A band assembly.
Spontaneous Ca2+ transients occur with a characteristic
mean frequency (Ferrari et al., 1996), raising the possibility that macromolecular assembly is encoded by this parameter. Because phosphorylation of the RLC by MLCK
occurs in seconds and dephosphorylation in minutes
(Somlyo and Somlyo, 1994
; Trybus, 1994
; Sobieszek et al.,
1997
), the RLC phosphorylation state has been suggested
to serve as a molecular form of short term "muscle memory" for twitch potentiation in adult skeletal muscle (Levine et al., 1996
). Since a myofibrillar phosphatase isoform
complexes directly with calmodulin and MLCK (Somlyo
and Somlyo, 1994
; Sobieszek et al., 1997
), periodic activation of MLCK by Ca2+ transients may be required to
maintain myosin in the conformation necessary for assembly, implying a threshold level of activity below which A
bands would fail to form.
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Footnotes |
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Received for publication 9 February 1998 and in revised form 1 May 1998.
Address all correspondence to Michael B. Ferrari, Ph.D., Department of Biology, University of California San Diego, La Jolla, CA 92093-0357. Tel.: (619) 534-2456. Fax: (619) 534-7309. E-mail: ferrari{at}biomail.ucsd.eduWe thank D. Dagan, T. Gomez, X. Gu, and M.-M. Poo for comments on the manuscript; A. Smith and A. Sanchez at Stanford's Beckman Center for peptides; P. Gallagher for MLCK antisera and A10 cell line lysate; and P. Kassner and W. Conroy for advice.
This work was supported by NIH grants to N.C. Spitzer and a Muscular Dystrophy Association postdoctoral research fellowship to M.B. Ferrari.
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Abbreviations used in this paper |
---|
Bis I, bisindolylmaleimide I; MLCK, myosin light chain kinase; MLCKi, myosin light chain kinase inhibitory peptide; pANT, antennapedia peptide; PKC, protein kinase C; PMA, phorbol 12-myristate, 13-acetate; RLC, regulatory light chain; RyR, ryanodine receptor Ca2+ release channel.
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References |
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