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Stanford University, Hopkins Marine Station, Pacific Grove, California 93950
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have cloned a group of cDNAs that encodes the skeletal ryanodine receptor isoform (RyR1) of fish from a blue marlin extraocular muscle library. The cDNAs encode a protein of 5,081 amino acids with a calculated molecular mass of 576,302 Da. The deduced amino acid sequence shows strong sequence identity to previously characterized RyR1 isoforms. An RNA probe derived from a clone of the full-length marlin RyR1 isoform hybridizes to RNA preparations from extraocular muscle and slow-twitch skeletal muscle but not to RNA preparations from fast-twitch skeletal or cardiac muscle. We have also isolated a partial RyR clone from marlin and toadfish fast-twitch muscles that shares 80% sequence identity with the corresponding region of the full-length RyR1 isoform, and a RNA probe derived from this clone hybridizes to RNA preparations from fast-twitch muscle but not to slow-twitch muscle preparations. Western blot analysis of slow-twitch muscles in fish indicates the presence of only a single high-molecular-mass RyR protein corresponding to RyR1. [3H]ryanodine binding assays revealed the fish slow-twitch muscle RyR1 had a greater sensitivity for Ca2+ than the fast-twitch muscle RyR1. The results indicate that, in fish muscle, fiber type-specific RyR1 isoforms are expressed and the two proteins are physiologically distinct.
fiber types
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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THE PROCESS OF excitation-contraction (EC) coupling in muscles involves the release of Ca2+ from sarcoplasmic reticulum (SR) stores. This release is mediated by the SR Ca2+ release channel, a large tetrameric protein complex of ~2.2 × 106 Da. The affinity of the SR Ca2+ release channel for the plant alkaloid ryanodine has provided a tool for the purification and biochemical characterization of this protein commonly referred to as the ryanodine receptor (RyR) (22, 23). Vertebrate RyRs identified to date are all similar-sized homotetrameric proteins composed of four polypeptide subunits with molecular masses of 500-600 kDa (37). mRNAs for the three known isoforms of the RyR gene family have been cloned and sequenced. The primary sequences for these isoforms were first characterized from mammalian tissues, including RyR1 from rabbit and human skeletal muscle (39, 42), RyR2 from rabbit cardiac muscle (29), and RyR3 from rabbit brain (20). Giannini et al. (19) extensively surveyed the expression of the three RyR isoforms in various murine tissues. With the use of both RNA antisense probes and isoform-specific antisera, a much wider tissue distribution of the mammalian isoforms of the RyRs emerged.
Three homologous isoforms of the RyR have also been identified in
nonmammalian tissues based on molecular, biochemical, immunologic, and
physiological results (1, 25, 28, 30, 31). In contrast to adult mammals
that predominantly express only the RyR1 isoform in skeletal muscle,
nonmammalian skeletal muscle expresses two isoforms, originally
described as 
- and 
-RyRs, due to unknown homologies to mammalian
isoforms (1, 25, 28). The cloning and characterization of the two
skeletal RyR isoforms from bullfrog have revealed the - and -RyRs
are homologous to the mammalian RyR1 and RyR3 gene products,
respectively (30, 31). Partial sequence data indicate that in lower
vertebrates the homolog of the RyR2 gene family is also present in
cardiac muscle (J. Keen and B. A. Block, unpublished data). In Western
blot analysis of nonmammalian skeletal muscles, RyR1 and RyR3 occur as
discrete high-molecular-weight bands, with RyR3 having a slightly
higher mobility. Exceptions to this expression pattern exist in certain nonmammalian muscles. In specialized muscle fibers of nonmammals, such
as the super-fast-contracting swim bladder muscle of toadfish, immunoblotting experiments have determined that RyR1 is the sole isoform expressed, making the muscle a pure source of the nonmammalian RyR1 isoform (3, 25). The ryanodine binding characteristics and
single-channel conductance of the fish RyR1 channel indicate it is the
functional homolog of the mammalian RyR1 protein (9, 32, 38). A second
exception to the two RyR isoform expression pattern of nonmammalian
skeletal muscles is the slow-twitch muscle fibers in fish. In a
preliminary report of the results in this paper, we demonstrated that
fish slow-twitch muscles do not express RyR3 (17).
Recent physiological studies of fish skeletal muscle fiber Ca2+ release kinetics indicate distinct Ca2+ transients are present in the slow- and fast-twitch muscles of fish (34). Studies in mammalian skeletal muscles also indicate that the mechanism of Ca2+ release differs between muscle fiber types (12, 13, 18, 36). The most prominent difference between fiber types is the time course of intracellular Ca2+ transients. In addition, different sensitivities of the RyR Ca2+ release mechanism (in fiber and SR vesicle preparations) to known modulators of the RyR channel, such as Ca2+, Mg2+, ATP, caffeine, doxorubicin, and ruthenium red, indicate distinct differences between slow- and fast-twitch vesicle preparations (24, 36). The molecular basis for the distinct Ca2+ transients has also been attributed to the presence of two isoforms of Ca2+-ATPase in skeletal muscles of tetrapods (5) as well as differences in the troponin off-rate of Ca2+ (34). In mammals and fish, slow fibers have longer Ca2+ transients and more sensitive force-pCa relationships. In this paper, we present evidence for two distinct fiber type-specific SR Ca2+ release channels in the skeletal muscles of fish that may also be contributing to the physiological differences between fiber types (34).
The discrete anatomic separation of fast- and slow-twitch muscle fibers in fish provides an unparalleled system for studying the biochemical and molecular components of different muscle fiber types. One of the richest sources of slow-twitch muscle is found in the large open ocean fishes (marlin and tunas) that use this muscle type to power endurance swimming across ocean basins as well as for thermogenic purposes (2). The slow-twitch (red) muscles of tuna and marlin are composed of 100% slow-twitch fibers, and the fast or white muscles are 99% pure sources of fast-twitch fibers (40). In this study, we constructed a cDNA library from the superior rectus muscle to characterize the message for the fish RyR1 isoform. Our interest in the superior rectus muscle stems from studies on the thermogenic potential of eye muscles in marlin (3). We report the cloning and characterization of a unique vertebrate RyR1 message from blue marlin (Makaira nigricans) eye muscle (a mixed fiber type muscle) that is also expressed in slow-twitch muscles of fish. We have also cloned and sequenced partial RyR1 messages from cDNA libraries derived from super-fast-contracting toadfish swim bladder muscle and fast-twitch muscle of marlin. Hybridization of RNA probes from these three sources reveals a fiber type-specific distribution of the fish RyR1 isoforms, indicating the presence of both slow and fast RyR1 messages. Ryanodine binding assays with heavy SR vesicles isolated from slow- and fast-twitch muscle fibers reveal distinct sensitivities of the Ca2+ dependence of the ryanodine receptor proteins.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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cDNA cloning and sequencing.
Total RNA was extracted from blue marlin (M. nigricans) tissues by either the guanidium
isothiocyanate/cesium chloride method of Chirgwin et al. (8) or by
using Tri-reagent (Molecular Research Center, Cincinnati, OH).
First-strand cDNA was synthesized from 10 µg total RNA using an
oligo(dT) primer. Reaction conditions for PCR included 200 ng template,
1 µM of each primer, 200 µM dNTPs, and 0.5 U Taq DNA polymerase
(Promega Biotech, Madison, WI). First-strand cDNA synthesized from
superior rectus muscle of blue marlin was used to amplify an ~750-bp
PCR product with primers RyR24
(5'-AAGGCATCAATGATCAGACCC-3') and RyR25
(5'-CTGTACATCACAGAGCAGCC-3'). The PCR product
corresponds to nucleotides 14056-14819 in the rabbit RyR1 open
reading frame (ORF) (42). This PCR product was used to screen a
commercially prepared oligo(dT)/random-primed cDNA library derived from
the superior rectus eye muscle of blue marlin (Stratagene, La Jolla,
CA). For the initial screening and all subsequent library screenings,
probes were labeled by random priming with
[-32P]dCTP
according to Feinberg and Vogelstein (15) or with the Ready-to-Go
random-priming kit (Pharmacia Biotech). The initial screening yielded
clone 
BMRR1 (ORF 14,044-15,332; Fig.
1).
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BMRR2 (ORF 5133-8127). For the
immunoscreening procedure, the library was plated at a density of
50,000 pfu/plate and incubated for 3.5 h at 42°C. The recombinant
clones were induced to express by placing nylon membranes impregnated
with 10 mM
isopropyl--D-thiogalacto-pyranoside
(Hybond-N, Amersham, Arlington Heights, IL) on the plates and
continuing incubation at 37°C for an additional 4 h. The membranes
were preblocked in 2% dried milk and 1× Tris-buffered saline and
0.2% Tween (TBST) for 1 h followed by a wash for 10 min in 1×
TBST. The primary antibody was diluted 1:1,000 in TBST and incubated
with the membranes for 2 h. After the primary antibody incubation, the
membranes were washed three times for 10 min each in 1× TBST. The
membranes were subsequently incubated with the secondary
antibody/alkaline phospatase conjugate diluted 1:1,000 (goat
anti-mouse) for 1 h. All incubations were performed at room
temperature.
After the secondary antibody incubation, the membranes were washed
three times for 10 min each in TBST. Positive clones were detected
colorimetrically with the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma Chemical, St. Louis, MO). The
human-specific RyR primer pair RR61FX
(5'-ATCTCTAGATCAAAAGCTGGGGAGCAGGAGGAG-3') and RR20XR
(5'-ACTTCTAGATTCAGAGCCCACAATGTCCTTGAG-3') amplified an approximate 1,200-bp PCR product from blue marlin first-strand cDNA
derived from the superior rectus muscle. This PCR product was
subsequently used to screen the random/oligo(dT)-primed superior rectus
muscle library, yielding two clones BMRR3 and BMRR4 (ORF 10,003-11,685). Screening with this PCR product also yielded
clones BMRR5 (ORF 11,700-13,685) and BMRR6 (ORF
11,375-11,725). The gap between clones BMRR4 and BMRR6 was
closed by PCR amplification from first-strand cDNA with primers
specific to the two clones. The PCR product pBMRR1 was cloned into the
vector pGEM and sequenced, which determined its identity to clones
BMRR4 and BMRR7 in overlapping regions. An approximate 800-bp PCR
product amplified from the 3'-terminus of clone BMRR2 was used
to screen the random/oligo(dT)-primed library, resulting in the
isolation of the clone BMRR7 (ORF 6800-8647). Clone BMRR8
(ORF 2879-6000) was isolated from the library after screening with
an ~800-bp PCR product derived from the 5'-end of clone
BMRR2. Two of the clones used in the assembly of the final RyR1
sequence were isolated from a primer extension library. An 18-mer
primer complementary to nucleotide residues 3030-3047 in the final
fish RyR1 ORF was used to prime first-strand synthesis from 2.5 µg
poly(A)+ RNA isolated with a
Poly(A) Quik mRNA isolation kit from the superior rectus muscle of
M. nigricans (Stratagene). After
second-strand synthesis was completed, the cDNAs were blunt ended by
adding 2 U Pfu DNA polymerase
according to the manufacturer's protocol (Stratagene).
EcoR I adaptors were blunt-end ligated
to the cDNAs and kinased using 10 U T4 polynucleotide kinase. Removal
of excess adaptors and size fractionation of the cDNAs were performed
by centrifugation through a S-500 Sephacryl column according to the manufacturer's instructions. The entire aliquot of the
size-fractionated cDNA was ligated to the ZAPII vector arms and
packaged using the Gigapack III Gold packaging extract. The primer
extension library was screened with a PCR product derived from the
5'-terminus of the BMRR8 cDNA clone. This screening yielded
clone BMRR9, which corresponds to nucleotides 985-3,191 in the
final RyR1 ORF. Clone BMRR9 was partially restriction mapped, and a
400-bp BamH I/Hind III restriction fragment from
the 5'-end of the clone was used to rescreen the primer extension
library. This yielded clone BMRR10, which corresponds to nucleotides
869-3,190 in the final ORF. The clone that codes for the
NH2 terminus of the fish RyR1 sequence was isolated from the original oligo(dT)/random-primed cDNA
library using an ~350-bp probe amplified from the 5'-end of
clone BMRR10. This screening yielded clone BMRR11, which contains
65 bp of 5'-untranslated sequence and extends to base 1,100 in
the final ORF. PCR-amplified regions of the 3'- and 5'-ends of clones BMRR7 and BMRR4 were radiolabeled and used to screen the oligo(dT)/random-primed library, resulting in the isolation of
clone BMRR12 corresponding to nucleotides 8,948-10,374 in the
final ORF. Rescreening of the library with a PCR-amplified region from
the 5'-end of clone BMRR12 yielded clone BMRR13, which
corresponds to nucleotides 6,941-8,690. The missing sequence between clones BMRR12 and BMRR13 was amplified from first-strand cDNA using a primer derived from the clones. The PCR clone was identical in sequence to the overlapping region of clone BMRR12 and
BMRR13. The insert from clone pBMRR2 was radiolabeled and used to
rescreen the oligo(dT)/random-primed library, which yielded two clones,
BMRR14 and BMRR15, which correspond to nucleotides 8,736-9,732 and 8,715-9,450, respectively, in the final ORF.
cDNA libraries were constructed from RNA isolated from marlin white
muscle RNA and toadfish swim bladder RNA using the ZAP kit of
Stratagene. Both libraries were screened with a radiolabeled probe
derived from the BMRR1 clone using the primers RyR24 and RyR25 (see
above). This screening yielded four clones from the toadfish swim
bladder (TFSB) library, named TFSB1 through TFSB4, and one clone
from the blue marlin white muscle (BMWM) library, named
BMWM1. The TFSB clones encompassed sequence corresponding to
nucleotides 13,695-14,950 of the blue marlin ORF, whereas the BMWM
clone contained sequence corrresponding to nucleotides
13,750-15,150.
Ribonuclease protection assays.
The RyR1-specific antisense probe was synthesized from a subcloned
region amplified from clone BMRR8 using U-strand primer RyR1Eco
(5'-TATGAATTCCTCAAGAAGTCTGCT-3') and L-strand primer RyRXho (5'-GATCTCGAGGTCGTCCCTGTCGTC-3'). The amplified
product was digested with the restriction enzymes
EcoR I and
Xho I and unidirectionally cloned into
Bluescript SK+ digested with EcoR I
and Xho I. The subcloned region
corresponds to nucleotides 4,075-4,315 in the blue marlin RyR1
ORF. The antisense probe was synthesized from the
EcoR I linearized clone with T7 RNA
polymerase according to the Ambion Maxiscript T7/T3 in vitro
transcription kit protocol (Ambion, Austin, TX). An antisense probe was
synthesized from a 375-bp region of clone TFSB1 using U-strand
primer (5'-AGGATTGAATTCATGAACTACTTG-3') and L-strand primer
(5'-AGGGAGCTCGAGGCAGTTGTATTC-3'). The PCR product was
digested with the restriction enzymes
EcoR I and
Xho I and unidirectionally cloned into
Bluescript SK+ digested with EcoR I
and Xho I. The subcloned region
corresponds to nucleotides 13,737-14,130 in the blue marlin RyR1
ORF. The antisense probe was synthesized from the
EcoR I linearized clone with T7 RNA
polymerase. Total RNA for the ribonuclease protection assays (RPA) was
prepared using Trisol reagent. The assay was performed according the
protocol of the Ambion Direct Protect RPA kit except that total RNA (20 µg) was used instead of tissue homogenates. All hybridizations were
performed at 37°C. Samples were separated on a 6% sequencing gel
that was dried and exposed to X-ray film for 24-72 h at
70°C with intensifying screens.
Heavy SR protein preparation. Approximately 10-25 g of blue marlin and tuna fast-twitch muscle, slow-twitch muscle, or toadfish swim bladder muscle were homogenized in 10 vol of homogenization buffer containing 300 mM sucrose, 5.0 mM Na2EGTA, 10.0 mM Na2EDTA, 20 mM K-PIPES, pH 7.3, 1.1 µM diisopropyl fluorophosphate, and various protease inhibitors using a Tekmar tissue homogenizer. Sodium pyrophosphate (25 mM) and 100 mM KCl were added to the homogenization, and the slurry was stirred on ice for 45 min to separate myofibrillar proteins from triads. The homogenate was centrifuged for 50 min at 100,000 g in a Ti50.2 Beckman rotor. The supernatant was discarded, and the pellet was resuspended in homogenization buffer and centrifuged at 2,000 g for 20 min in a Sorval SS34 rotor. The supernatant was passed through two layers of cheese cloth, and the crude microsomes were pelleted by centrifugation at 100,000 g for 50 min. The pellets were resuspended in 300 mM sucrose and 5 mM K-PIPES, pH 7.0. This material was layered onto discontinuous sucrose gradients (6 ml 20%, 8 ml 30%, 8 ml 36%, and 4 ml 45%) containing 0.4 M KCl, 0.1 mM Na2EGTA, 0.1 mM CaCl2, and 5.0 mM K-PIPES, pH 6.8, and centrifuged 16 h at 25,000 rpm in Beckman SW25 rotor. Heavy SR membrane fractions were collected by aspiration from the 36%-45% interface, diluted with ice-cold water, and pelleted at 100,000 g in a Beckman Ti50.2 rotor. Pelleted membranes were resuspended in 300 mM sucrose and 5 mM K-PIPES, pH 7.0, and frozen in liquid nitrogen until use.
[3H]ryanodine binding
assays.
Heavy SR vesicles were incubated for 2 h at 30°C in binding medium
containing 10 nM
[3H]ryanodine, 0.2 M
KCl, 1.0 mM 
-methyleneadenosine 5'-triphosphate, 1.0 mM
Na2EGTA, and 20 mM
K-PIPES, pH 7.1. The free
Ca2+ concentration of the medium
was adjusted to 0.1-1,000 µM by the addition of
CaCl2 according to the affinity
constants of Fabiato (14). After incubation, the samples were filtered
onto Whatman GF/B glass fiber filters and washed twice with 5 ml
ice-cold distilled water. Nonspecific binding was measured in the
presence of 10 µM unlabeled ryanodine and was subtracted
from each sample.
Sequence and phylogenetic analysis. For the determination of putative transmembrane regions, the protein structure was predicted using the PredictProtein server (35). Sequences were submitted using the PredictProtein interactive Web site (http://www.embl-heidelberg.de/predictprotein). Transmembrane predictions were made using a neural network method with a >95% expected accuracy per residue.
The deduced amino acid sequences for the fish RyR1 sequences were aligned to all published RyR amino acid sequences using the program CLUSTAL W (21). A phylogenetic tree based on parsimony was generated using the ProtPars algorithm of PHYLIP (16). Confidence values on the major nodes of the tree were calculated by generating 500 replicate data sets with replacement using the SeqBoot program of PHYLIP. The multiple data sets were used as the input for the ProtPars program.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Sequence determination of a full-length RyR1 isoform in fish. Screening of an oligo(dT)/random-primed library and a specific primer extension library with DNA and antibody probes resulted in the identification of a series of overlapping clones (Fig. 1). Compilation of the cDNA clones resulted in a 16,313-bp contiguous sequence. The contiguous sequence codes for an ORF of 15,243 bp with the initiation methionine at position 66 and the termination codon (TAG) at position 15,309. A termination codon TAA is found 15 bp upstream of the initiator methionine. The 3'-untranslated region is 1,002 nucleotides long. Polyadenylation signals with the motif AATAAA were located at 961, 892, and 247 bases upstream from the 3'-terminus. The complete fish RyR cDNA sequence encodes a protein of 5,081 amino acids with a deduced molecular mass of 576,302 Da, including the initiator methionine. A multiple alignment of the deduced amino acid sequence to RyR1 sequences of frog, rabbit, and human illustrates that the four sequences share large regions of sequence identity especially toward the highly conserved COOH terminus of the molecule (Fig. 2. The fish sequence is longer than the other three vertebrate RyR1 sequences, which is largely accounted for by two insertions, one of 30 amino acids from amino acid 1,851-1,880 and one of 25 amino acids from amino acids 1,926-1,950. The overall sequence identities between the fish RyR1 sequence and published sequences are given in Table 1. The deduced amino acid sequence from fish shares the greatest sequence identity with the frog RyR1 sequence (77%) and is 72 and 73% identical in pairwise comparisons with human and rabbit RyR1 amino acid sequences, respectively.
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Identification of fast-twitch muscle specific RyR1 isoforms.
To determine if fish express fiber type-specific RyR1 isoforms, we
screened cDNA libraries derived from toadfish swim bladder (RyR1 only
muscle) and blue marlin fast-twitch muscles (RyR1 and RyR3) (25). Both
libraries were screened with a radiolabeled probe amplified from the
BMRR1 clone. This screening yielded four clones from the TFSB
library named TFSB1 through TFSB4 and one clone from the BMWM
library named BMWM1. The TFSB clones generated a contiguous sequence
corresponding to nucleotides 13,695-14,950 of the blue marlin eye
muscle RyR ORF, whereas the BMWM clone contained sequence corresponding
to nucleotides 13,750-15,150.
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TFSB1 (Fig. 7). The RNA probe
hybridized to RNA preparations from toadfish swim bladder, toadfish
fast-twitch muscles, marlin fast-twitch muscle, and tuna fast-twitch
muscle. Importantly, the message could not be detected by
hybridization, even after long exposures, in marlin or tuna slow-twitch
muscle. An RNA probe constructed from the blue marlin fast-twitch
muscle clone also hybridized to RNA isolated from the fast-twitch
muscles but not the slow-twitch muscles (data not shown).
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Ryanodine binding and Western blot analysis. The previous results suggest that two distinct RyR1 isoforms are expressed in fish skeletal muscle in a fiber-type specific manner. [3H]ryanodine binding was performed to characterize the properties of the RyR isoforms in fish fast- and slow-twitch muscles. [3H]ryanodine binding to RyR1 in mammalian skeletal muscle SR vesicles displays a classic bell-shaped Ca2+ dependency, with activation occurring at micromolar Ca2+ concentrations and inactivation occurring at millimolar Ca2+ concentrations. [3H]ryanodine binding to SR preparations from marlin fast-twitch muscle exhibited this bell-shaped Ca2+ dependency with a peak at pCa 4 (Fig. 8A). The SR preparation from marlin slow-twitch muscle also exhibited a bell-shaped Ca2+ dependency but with the peak shifted to a lower [Ca2+] of pCa 5 (Fig. 8B). Additional ryanodine binding assays with SR preparations from tuna fast-twitch muscle and tuna slow-twitch muscle confirmed the marlin results (Fig. 8, D and E). The data from the binding curves in Fig. 8 were normalized to show the amount of [3H]ryanodine binding relative to the peak for each curve (Fig. 9). The left shift in the Ca2+ sensitivity for the slow-twitch preparations is clearly evident. Western blot analysis using the antibody C010, which reacts against an epitope common to all RyRs (25), detected two high-molecular-weight bands in the fast-twitch (white) muscle preparations previously identified as RyR1 and RyR3 (25). Suprisingly, only one band, with a mobility similar to the RyR1 protein of fast-twitch muscles, was found in the slow-twitch muscle SR preparations (Fig. 10). Thus the properties examined in the [3H]ryanodine binding assays can be attributed to this one RyR protein expressed in the slow-twitch preparations.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The results of this study suggest that fast- and slow-twitch muscles of fish express specific RyR isoforms. The full-length RyR1 sequence presented here represents a novel vertebrate isoform of the RyR1 gene family. The cDNA isolated from the superior rectus muscle library encodes a deduced amino acid sequence that is 77% identical in pairwise comparison to the frog RyR1 isoform. The calculated molecular mass of 576 kDa is also similar to the RyR1 isoforms of mammals and amphibians (31, 39, 42). Molecular phylogenetic analyses also confirm that the cDNA codes for a RyR isoform that is most closely related to the RyR1 isoforms (Fig. 3).
Hybridization of an antisense RNA probe was performed to resolve the tissue distribution of the fish eye muscle RyR1 isoform. The message for the fish RyR1 isoform is preferentially expressed in the slow-twitch (red) muscle of fish (Fig. 4). Surprisingly, this RyR1 message is not expressed in fast-twitch muscles of three fish species examined (marlin, tuna, and toadfish). The message can only be detected in minor abundance if exposure times are significantly increased, and this most likely corresponds to the presence of a small number of slow-twitch fibers in these muscles. The message is detected in the mixed fiber type superior rectus muscle (40) from where the library was originally constructed. The full-length cDNA we have cloned and sequenced is designated RyR1 slow. The expression results prompted us to determine if a second, fast-twitch muscle RyR1 isoform may also be expressed in fish muscles. We constructed and screened cDNA libraries derived from marlin fast-twitch muscle and toadfish swim bladder muscle and were able to obtain partial RyR sequences from both tissues. Previous expression studies based on the presence of isoforms of the Ca2+-ATPase (SERCA1 and SERCA2) have shown the toadfish swim bladder muscle is composed of only fast-twitch fibers (40). The toadfish swim bladder muscle and marlin fast-twitch muscle sequences share high identity with the RyR1 gene family and are similar but not identical to the RyR sequence derived from the extraocular eye muscle library. Phylogenetic analyses determined that these partial sequences are closely related to the fish slow-twitch isoforms but significantly distinct. Importantly, the marlin fast-twitch muscle and toadfish swim bladder muscle sequences are more closely related to each other than either is to the marlin slow-twitch sequence (Fig. 6). A probe derived from the toadfish swim bladder muscle RyR1 clone hybridizes to messages in swim bladder muscle and fast-twitch muscle fibers of marlin, toadfish, and tuna, but not to slow-twitch muscle from marlin and tuna (Fig. 7). These results indicate that fish express fiber type-specific RyR1 isoforms.
Although several splice variants of the mammalian RyR1 message have been described (33, 43), the current study represents the first evidence of unique RyR1 gene products in fast- and slow-twitch muscle fibers. The two partial sequences isolated from the fast-twitch fish muscles share significant amino acid substitutions (shared derived characters) when compared with the slow-twitch isoform. This indicates that within the channel region for which the limited comparative sequence data exist, there are significant differences between the fast and slow isoforms that represent evolutionary divergences in the RyR1 isoforms. The fact that fiber type-specific RyR1 isoforms have not been characterized in mammals or amphibians may be associated with a loss of one of the isoforms in higher vertebrates, but it could also be attributed to the bias of selecting fast-twitch muscles for the construction of cDNA libraries, since these muscles contain a high content of SR (26, 31, 39, 42). Londraville et al. (unpublished data) have recently shown that SERCA1b, a neonatal form of the Ca2+-ATPase in mammals, is expressed in adult extraocular muscles of fish and birds but not in extraocular muscles of adult mammals. Thus distinct expression patterns in the SR proteins of lower vertebrates may be a common finding once investigated in further detail.
The anatomic arrangement of fish muscles provides the tool for separating the pure slow-twitch from fast-twitch muscle, making the expression and binding studies possible. To determine whether the expression of unique RyR1 isoforms in fast- and slow-twitch muscles of fish results in functional differences, we assayed SR fractions from the different muscle fiber types for their affinity for [3H]ryanodine. Ryanodine binding in skeletal muscle SR preparations is typically activated by submicromolar Ca2+ concentrations and inhibited by millimolar Ca2+ concentrations. [3H]ryanodine binding to both fast- and slow-twitch SR preparations from fish exhibited the classic bell-shaped dependence on Ca2+ concentration that is characteristic of skeletal RyR isoforms. The pCa for peak binding was, however, different for the two muscle fiber types. The fast-twitch muscle showed peak binding at pCa 4, whereas the slow-twitch muscle fibers exhibited peak binding at pCa 5 (Figs. 8 and 9). This indicates that the RyR isoforms of slow-twitch muscle fibers have a significantly lower threshold for Ca2+ activation.
Nonmammalian skeletal muscles typically coexpress both the RyR1 and RyR3 isoforms (25). Immunoblot analysis of SR preparations using an antisera that recognizes an epitope common to all RyR isforms revealed that although two isoforms can be detected in marlin fast-twitch muscle (RyR1 and RyR3), the slow-twitch muscle SR preparation only expresses a single RyR isoform (Fig. 9). The mobility of the single band on the protein gels and recognition by the C010 antibody along with the RNase hybridization results with the probe generated from the full-length cDNA for RyR1 slow isoform indicate this is the RyR1 slow protein. In addition, we have generated an RyR1-specific antibody that recognizes only the RyR1 protein in fast- and slow-twitch muscles of marlin (17). Because of this result, the differences observed between the fast-twitch and slow-twitch SR preparations for ryanodine binding could be ascribed to the influence of the coexpression of the RyR1 and RyR3 isoforms. However, the toadfish swim bladder muscle is known to be composed of a homogeneous fiber type that only expresses the RyR1 isoform (25). A SR preparation derived from toadfish swim bladder muscle also exhibits the bell-shaped dependency on Ca2+ concentration with a peak binding at pCa 4, similar to the fast-twitch white muscle preparations of marlin and tuna (Fig. 8E). Therefore, the shift in peak binding for ryanodine binding for the slow-twitch muscle preparation cannot be attributed to the absence of the RyR3 isoform in the preparation. These results were demonstrated for two fish species from which significant quantities of slow-twitch muscle can be isolated (tuna and marlin). Toadfish have extremely small amounts of slow-twitch muscle fibers, which limits the ability to do protein analyses.
The absence of the RyR3 protein as revealed by Western blots raises interesting questions. It may be due to a reduced need to amplify the RyR1 signal as has been proposed in the two-component model for Ca2+ release (27) or a property of the RyR1 slow isoform that necessitates building triads with only this protein. It is possible that the slow-twitch muscle fibers in fish operate in vivo at lower thresholds of Ca2+ than fast-twitch fibers, possibly necessitating the construction of the triad with RyR isoforms that share similar properties (low threshold for activation via Ca2+). If triads were built with a mixture of RyR isoforms that had different sensitivities to Ca2+ for opening and closing, Ca2+ release may not be as efficient.
Previous studies have described physiological differences in the mechanism of EC coupling between fast- and slow-twitch muscles. Salviati and Volpe (36) compared the kinetics of Ca2+ release from rabbit skinned fast and slow-twitch fibers, determining that the Ca2+ release channels of both tissue types respond to known modulatory agents but show different sensitivities. The SR preparations of slow-twitch muscle fibers had a lower threshold for caffeine, whereas the fast-twitch SR preparations were found to be more sensitive to ryanodine. Lee et al. (24) further showed with planar lipid bilayer recordings that rat fast- and slow-twitch muscle Ca2+ release channels have different rates of initial Ca2+ release and mean channel closed times. The Ca2+ released from the slow-twitch vesicles was 28% less than from fast-twitch SR vesicles. Recently, Delbono and Meissner (11) compared the intracellular transients in rat fast- and slow-twitch fibers using the low-affinity Ca2+ indicator Mag-fura 2 and demonstrated that rat fast-twitch muscles removed myoplasmic Ca2+ faster than slow-twitch muscle. They also determined with binding experiments that slow-twitch muscles have a lower ratio of dihydropyridine receptors to RyRs, concluding that a lower number of the RyRs in slow-twitch muscle are directly controlled by the dihydropyridine receptor. Fish muscle fibers also demonstrate markedly different Ca2+ transients (34). In fish, the observed differences in Ca2+ transients and force generation by specific muscle fibers are most likely because of several molecular and biochemical modifications of SR and myofibrillar proteins, including the expression of fiber type-specific skeletal RyR isoforms.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. MacLennan for contribution of several primers.
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FOOTNOTES |
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The research was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-40246 and National Science Foundation Grant IBN-9507499 to B. Block. J. Franck was supported by a National Sciences and Engineering Research Council Postdoctoral Fellowship, J. Morrissette by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-08425, and J. Keen by the British Columbia Heart and Stroke Foundation.
The blue marlin RyR1 slow accession number in GenBank is U97329.
Present addresses: J. P. C. Franck, Dept. of Biology, Occidental College, 1600 Campus Rd., Los Angeles, CA 90041; R. L. Londraville, Dept. of Biology, Univ. of Akron, Akron, OH 44325-3908.
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. §1734 solely to indicate this fact.
Address for reprint requests: B. A. Block, Stanford University, Hopkins Marine Station, Oceanview Boulevard, Pacific Grove, CA 93950.
Received 16 March 1998; accepted in final form 23 April 1998.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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, Tuna slow-twich red muscle; 
,
blue marlin slow-twitch red muscle; 
, tuna fast-twitch white muscle;
, blue marlin fast-twitch white muscle; 
, toadfish swim bladder
muscle.
