(Received for publication, August 3, 1995; and in revised form, September 22, 1995)
From the
Triadin is an intrinsic membrane protein first identified in the skeletal muscle junctional sarcoplasmic reticulum and is considered to play an important role in excitation-contraction coupling. Using polyclonal antibodies to skeletal muscle triadin, we have identified and characterized three isoforms in rabbit cardiac muscle. The cDNAs encoding these three isoforms of triadin have been isolated by reverse transcription-polymerase chain reaction and cDNA library screening. The deduced amino acid sequences show that these proteins are identical in their N-terminal sequences, whereas the C-terminal sequences are distinct from each other and from that of skeletal muscle triadin. Based upon both the amino acid sequences and biochemical analysis, all three triadin isoforms share similar membrane topology with skeletal muscle triadin. Immunofluorescence staining of rabbit cardiac muscle with antibodies purified from the homologous region of triadin shows that cardiac triadin is primarily confined to the I-band region of cardiac myocytes, where the junctional and corbular sarcoplasmic reticulum is located. Furthermore, we demonstrate that the conserved region of the luminal domain of triadin is able to bind both the ryanodine receptor and calsequestrin in cardiac muscle. These results suggest that triadin colocalizes with and binds to the ryanodine receptor and calsequestrin and carries out a function in the lumen of the junctional sarcoplasmic reticulum that is important for both skeletal and cardiac muscle excitation-contraction coupling.
Ca release from the sarcoplasmic reticulum of
skeletal and cardiac muscle is regulated by similar but distinct
mechanisms(1, 2, 3) . In cardiac muscle,
depolarization leads to the opening of voltage-gated Ca
channels. Ca
influx through L-type
Ca
channels (the dihydropyridine receptor) triggers
the opening of the ryanodine receptor/Ca
release
channel in the sarcoplasmic reticulum. However, in skeletal muscle,
entry of external Ca
is not needed for this signal
transduction process. The skeletal muscle dihydropyridine receptor
interacts either directly or indirectly with the ryanodine receptor,
thereby activating the Ca
release channel without a
requirement for extracellular calcium. Despite this difference, many
factors that modulate the channel properties of the skeletal muscle
ryanodine receptor also affect the cardiac ryanodine receptor in a
similar fashion(4, 5) . Identification of protein
components in the junctional sarcoplasmic reticulum is fundamental to
our understanding of the mechanisms of Ca
storage and
release in muscle cells. So far, the major components of the
excitation-contraction coupling, such as the ryanodine receptor, the
dihydropyridine receptor, and calsequestrin, have been extensively
studied in skeletal muscle, and their counterparts have later been
identified and characterized in cardiac muscle.
Triadin is an
intrinsic membrane protein originally identified in skeletal
muscle(6, 7, 8) . It is specifically enriched
in the junctional sarcoplasmic reticulum, where it colocalizes with the
ryanodine receptor/Ca release
channel(6, 8) . In addition, triadin probably forms
multimers in the lumen of the sarcoplasmic reticulum through disulfide
bonds(6, 7, 8) . Triadin was previously
proposed to bind the ryanodine receptor and the dihydropyridine
receptor and to serve as the ``linking protein'' that
mediates the signal transduction process between these two
Ca
channels in skeletal muscle(8) . Recently,
triadin has been found to interact with both the ryanodine receptor and
calsequestrin in the lumen of the skeletal muscle sarcoplasmic
reticulum(9) . These results (9) along with membrane
topology analysis (7, 23) suggest that triadin
anchors calsequestrin to the junctional face membrane near the
sarcoplasmic reticulum ``foot'' ryanodine receptor and is
probably involved in the functional coupling between the Ca
release channel and the intraluminal calcium-binding protein
calsequestrin(10, 11, 12, 13) .
Recent reports have described the possible existence of triadin in cardiac muscle(14, 15, 16, 17) . To gain further insight into the function of triadin and to examine the molecular differences between skeletal and cardiac muscle triadin, we have identified and biochemically characterized three cardiac triadin isoforms and subsequently cloned the cDNAs encoding these proteins. Immunofluorescence staining indicates that these cardiac triadin isoforms are primarily confined to the junctional and corbular sarcoplasmic reticulum. In addition, using an affinity binding assay, we found that the conserved region of the luminal domain of triadin is able to interact with both the ryanodine receptor and calsequestrin in the lumen of the cardiac muscle junctional sarcoplasmic reticulum. These results suggest that triadin does not carry out a skeletal muscle-specific function. Instead, it probably plays an important role in both skeletal and cardiac muscle excitation-contraction coupling.
Figure 1: Identification of cardiac triadin isoforms by Western blot analysis. Polyclonal antibodies generated from guinea pigs (GP57 and GP58) and sheep (Sh33) and a monoclonal antibody (IIG12) were used to detect triadin in 100 µg of rabbit cardiac microsomes using Western blot analysis. The molecular mass standards (in kilodaltons) are indicated on the left.
Figure 2: Vesicle protection assay of cardiac triadin isoforms. Rabbit cardiac muscle microsomes were treated with a 1:50 ratio of trypsin in the presence or absence of 0.3% CHAPS for 15 min at 37 °C. The reactions were stopped with 2 mM phenylmethylsulfonyl fluoride. The protein samples were separated by 3-15% SDS-PAGE and transferred to nitrocellulose. The immunoblot was stained with polyclonal antibody GP58. The molecular mass standards (in kilodaltons) are indicated on the left.
Figure 3:
Effects of sulfhydryl agents on the
migration of triadin on SDS-PAGE. Rabbit skeletal muscle triads (left) and cardiac muscle microsomes (right) were run
on 3-15% SDS-polyacrylamide gels in the presence of either 0.1%
-mercaptoethanol (
-MER.) or 5 mMN-ethylmaleimide (NEM). After transferring to
nitrocellulose, the blots were stained with antibody IIG12 (left) or GP58 (right) at a 1:1000 dilution. The
molecular masses (in kilodaltons) are indicated on the
left.
Figure 4: Isolation of cardiac muscle triadin DNA sequence by RT-PCR. Primers corresponding to the skeletal muscle triadin sequences were used to amplify the cardiac triadin sequence from rabbit cardiac muscle RNA (see ``Experimental Procedures''). A fragment of 845 base pairs were generated by primers F1 and R2 (lane 2). However, no fragment was amplified using primers F1 and R1 (lane 1).
This 845-bp fragment was then used as probe
for hybridization screening of a ZAPII cDNA library constructed
from poly(A) RNA isolated from an adult rabbit cardiac muscle. Two
clones that contain open reading frames were named cardiac triadin 1
(CT1) and cardiac triadin 2 (CT2). Their nucleotide sequences and
translations are shown in Fig. 5(A and B).
Both clones share the same 5`-sequences with skeletal muscle triadin
until nucleotide 793 and then diverge with the remainder of their
sequences. Because of their identity at the 5`-sequences, the
predictions for the initiation codons of the cardiac isoforms are the
same as those for the skeletal sequence(7) . The predicted
molecular masses of CT1 and CT2 are 32,097 and 34,596 Da, respectively.
These probably correspond to the 35- and 40-kDa doublets detected on
the Western blots (Fig. 1). The 35-kDa isoform was solubilized
with 2% Triton X-100 and purified from the cardiac sarcoplasmic
reticulum using a phosphocellulose column. Three peptide fragments were
generated by endoproteinase Asp-N digestion and isolated by high
pressure liquid chromatography. The obtained peptide sequences
EGNASTTTTV, DPLKLV, and EKPERKI totally match the deduced triadin
sequence (Fig. 5A).
Figure 5: cDNA sequences and protein translations of cardiac triadin 1 (A), cardiac triadin 2 (B), and cardiac triadin 3 (C). The double underlined sequences denote the putative transmembrane-spanning domain. The single underlined amino acids were confirmed by peptide sequencing. The boldface letters indicate the amino acid residues that are unique to the cardiac triadin isoforms.
The homologous region of the
small triadin isoforms ends at nucleotide 793. However, the RT-PCR
product is 845 bp. The 52-bp sequence at the 3`-end is identical to
that of the skeletal sequence, but differs from CT1 and CT2. This
sequence probably represents part of the 92-kDa cardiac triadin (CT3)
sequence. A forward primer corresponding to nucleotides 795-815
was synthesized (TCAGTATGCATTCTGTCGATA). Using this primer together
with pBluescript primer KS, we were able to amplify the 3`-sequence
from the ZAPII library to obtain the full-length open reading
frame of CT3. Fig. 5C shows the nucleotide sequence of
CT3 and its translation. In comparison with skeletal muscle triadin (Fig. 6), except for the deletion of GCA from positions 991 to
993, CT3 has the same sequence from nucleotides 1 to 1932. The rest of
the 3`-sequence is totally different. The translated protein therefore
has a unique C-terminal sequence of 23 amino acids. Fig. 6shows
the alignment of the three cardiac triadin isoforms together with the
skeletal muscle sequence. Since the conserved regions of these triadin
sequences are the same at the nucleotide level, these isoforms are
probably the result of alternative splicing of the same gene.
Hydrophobicity analysis of the proteins was conducted according to the
method of Kyte and Doolittle (20) using PC/GENE software (Fig. 7). The hydrophobicity plots suggest that all three
proteins share similar membrane topology with their skeletal muscle
counterpart, although their luminal segments have different lengths.
Figure 6: Protein sequence comparison of skeletal and cardiac muscle triadin isoforms. Amino acid sequences of skeletal muscle triadin (SKM) and cardiac triadin isoforms CT1, CT2, and CT3 are aligned. The residues that are identical in all the isoforms are indicated by asterisks; the conserved sequences between skeletal muscle and CT3 are denoted by colons.
Figure 7: Hydrophobicity analysis of cardiac triadin isoforms. The hydrophobicities of the three cardiac triadin isoforms (CT1, CT2, and CT3) were analyzed by the method of Kyte and Doolittle (20) using a window size of 19 residues. Abscissa values represent amino acid numbers, while ordinate values indicate the relative hydrophobicity.
Figure 8: Localization of triadin in cryosections of adult rabbit atrial and ventricular muscle by immunofluorescence labeling. Cryosections of atrial (a) and ventricular (b and c) muscle tissues were immunofluorescently labeled with affinity-purified antibodies to H-triadin (GP58) and imaged by confocal microscopy. Comparison of the immunofluorescence staining pattern in a longitudinal section of rabbit papillary muscle (c) with the position of the A- and I-bands in the same field imaged by phase-contrast microscopy (d) showed that transversely oriented rows of discrete fluorescent foci (c, white arrows) were localized at the center of the I-band region (d, white arrows), where most of the junctional and corbular sarcoplasmic reticulum is localized in mammalian papillary myofibers. Note that the intensity of labeling for triadin was very high in atrial (m; a) and ventricular (m; b and c) myofibers, while the intensity of labeling in endothelial cells (E; a) and arterial smooth muscle cells (sm; a) was only marginally higher than that of the background.
Regarding the subcellular distribution of triadin, confocal imaging of longitudinal cryosections from rabbit papillary muscle shows that specific labeling was mainly confined to parallel strands oriented transversely to the longitudinal axis of the fibers (Fig. 8, b and c). These strands were frequently resolved into discrete foci (Fig. 8c, white arrows). Phase-contrast imaging of the same field (Fig. 8d) shows that the discrete fluorescent foci were primarily localized to the center of the I-band region (Fig. 8d, white arrows), where most of the internal junctional and corbular sarcoplasmic reticulum is localized in mammalian myofibers.
Figure 9:
The homologous region of cardiac triadin
binds calsequestrin and the ryanodine receptor in cardiac muscle
microsomes. A, binding of cardiac muscle calsequestrin to
triadin fusion proteins. The calsequestrin in the starting material (Start), in the flow-through material (Void), and
eluted from the fusion protein-Sepharose column (Elution) was
detected using polyclonal antibody Rabbit H at a 1:100 dilution. B, binding of triadin fusion proteins to
[H]ryanodine-labeled receptors in rabbit cardiac
muscle microsomes. As a positive control, anti-ryanodine receptor
polyclonal antibody (RyR-Ab)-Sepharose was also used in the
binding assay.
To examine
whether H-triadin interacts with the ryanodine receptor, we prelabeled
the solubilized cardiac muscle microsomes with
[H]ryanodine. Triadin-GST fusion
protein-Sepharose was incubated with the radiolabeled material, and the
amount of bound receptor was determined by counting the
[
H]ryanodine on the Sepharose. As shown in Fig. 9B, both L-triadin and H-triadin bound the labeled
receptor, whereas C-triadin did not bind to the receptor. As a positive
control, the ryanodine receptor polyclonal sheep antibody (6) Sepharose was capable of binding to the solubilized
ryanodine receptor.
We have previously cloned skeletal muscle triadin and found that triadin was present only in skeletal muscle by Northern and Western blot analyses(7) . This suggested that triadin carries out a function that is specific to skeletal muscle excitation-contraction coupling. Recently, however, several reports have suggested the existence of triadin in cardiac muscle(14, 15, 16, 17) . Peng et al.(15) were able to detect several messages of triadin in cardiac muscle using Northern blot analysis. Notably, the messages could be identified using a probe corresponding to the 5`-fragment (but not the 3`-fragment) of skeletal muscle triadin. This result suggests that skeletal and cardiac muscle triadin isoforms share similar sequences at the N-terminal region, but differ at their C termini.
Using polyclonal antibodies, we demonstrate the existence of three isoforms of triadin in cardiac muscle microsomes (Fig. 1). One migrates at a molecular mass of 92 kDa, slightly smaller than the skeletal muscle counterpart of 95 kDa. The other two form doublets of 35 and 40 kDa, respectively. Interestingly, none of the proteins could be detected by monoclonal antibody IIG12, which was used to isolate the skeletal muscle triadin cDNA(7) . In fact, none of the monoclonal antibodies that were used by Knudson et al.(6, 7) to characterize skeletal muscle triadin were able to recognize the cardiac muscle triadin isoforms by either Western blot analysis or immunocytochemical staining (data not shown). The antigenic epitopes for these monoclonal antibodies have now been mapped to the C-terminal region of skeletal muscle triadin (data not shown), further supporting the prediction that the triadin isoforms differ at their C termini.
By RT-PCR and library screening, we have obtained cardiac triadin sequences (Fig. 5). The alignment of triadin sequences is shown in Fig. 6. These sequences are the same until nucleotide 793. For the long cardiac triadin isoform CT3 (Fig. 5C), except for the deletion of the trinucleotide GCA at positions 991-993, the nucleotide sequence is identical to that of skeletal muscle triadin from nucleotides 1 to 1932. The rest of the sequence is different. As a result of the GCA deletion, an alanine residue at position 331 is missing. The translated protein is therefore basically homologous to skeletal muscle triadin amino acids 1-665, but has a unique C terminus of 23 amino acids. The cardiac triadin isoform CT3 is 39 amino acids shorter than its skeletal counterpart, which explains why it has a slightly smaller molecular mass than skeletal muscle triadin as detected by Western blot analysis.
In skeletal muscle triadin, there are two cysteine residues at
positions 270 and 671 in the proposed luminal segment. These two
residues are believed to be responsible for the disulfide-linked
homomultimeric structure of triadin(7) . Interestingly, for
CT3, there is only one cysteine residue (position 270) in the luminal
segment. Fig. 3demonstrates that CT3 only migrates as a single
band of 92 kDa in the presence of N-ethylmaleimide.
Therefore, CT3 does not form multimeric complexes through disulfide
bonds. The lack of a cysteine residue might affect the formation of
multimeric structures of cardiac triadin.
Based on hydrophobicity analysis by the method of Kyte and Doolittle (20) , each of the three cardiac triadin isoforms contains a single transmembrane domain that separates it into cytoplasmic and luminal segments (Fig. 7). Since cardiac triadin isoforms are homologous to skeletal muscle triadin at their N-terminal portions, previous analysis of this portion of the sequences (7) applies to these cardiac proteins. Like their skeletal muscle counterpart, only the N-terminal 47 amino acids are cytoplasmic, with the bulk of the proteins located in the lumen of the sarcoplasmic reticulum. This prediction is consistent with the results of the vesicle protection assay (Fig. 2).
The cellular and subcellular distribution of triadin in cardiac muscle was also examined by immunofluorescence staining (Fig. 8). Specific staining was primarily detected in cardiac myocytes where the labeling of triadin corresponds very closely to that of the ryanodine receptor and calsequestrin, which were previously demonstrated to be localized to the junctional and corbular sarcoplasmic reticulum in cardiac myocytes(24, 25, 27, 28) . The subcellular distribution of triadin in cardiac muscle in this study is very similar to that reported by Brandt et al.(16) and Carl et al.(17) using a monoclonal antibody that recognizes both skeletal and cardiac muscle triadin.
We also examined the possible interaction of triadin with
the ryanodine receptor and calsequestrin in cardiac muscle. As shown in Fig. 9, the homologous region of the triadin luminal segment
(H-triadin) was able to bind [H]ryanodine-labeled
cardiac receptor and cardiac calsequestrin.
Triadin was originally found only in skeletal muscle(6, 7) . It was therefore proposed that triadin carries out a function that is specific to skeletal muscle excitation-contraction coupling(6, 7) . Recently, we found that triadin interacts with the ryanodine receptor and calsequestrin in the lumen of the skeletal muscle sarcoplasmic reticulum(9) . Triadin may be the transmembrane protein that anchors calsequestrin to the junctional sarcoplasmic reticulum near the ``sarcoplasmic reticulum foot'' ryanodine receptor and thus may be involved in the functional coupling between these two proteins (10, 11, 12, 13) . In cardiac muscle, calsequestrin and the ryanodine receptor are localized to the junctional and corbular sarcoplasmic reticulum membrane. If triadin is the anchoring protein for calsequestrin in skeletal muscle, there should be triadin homologs in cardiac muscle. In this study, we report the identification, molecular cloning, immunofluorescent localization, and biochemical characterization of three isoforms of triadin in rabbit cardiac muscle. We find that cardiac triadin isoforms share a large region of sequence identity with their skeletal muscle counterpart. In addition, their similar membrane topologies and patterns of molecular interactions suggest that triadin plays a role that is important to both skeletal and cardiac muscle excitation-contraction coupling.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31540[GenBank], U31555[GenBank], and U34201[GenBank].