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INTRODUCTION |
The sarcoplasmic reticulum
(SR)1 plays a central role in
the regulation of the cytosolic Ca2+ concentration and for
excitation-contraction coupling in cardiac muscle. Ca2+
release from the junctional SR increases the cytosolic Ca2+
concentration, which leads to initiation of the heartbeat by activation
of the contractile mechanism. The SR-Ca2+ATPase (SERCA2a)
in the free SR takes up Ca2+, thereby lowering the
cytosolic Ca2+ concentration and promoting muscle
relaxation. Ca2+ release in myocardium is controlled by a
complex of proteins localized to the junctional SR. These proteins
include (a) the Ca2+ release channel or
ryanodine receptor, which forms the foot structure on the cytoplasmic
surface of the junctional SR membrane; (b) the 55-kDa high
capacity Ca2+-binding protein calsequestrin, located in the
lumen of the junctional SR; and (c) the junctional SR
transmembrane proteins junctin and triadin, which have been proposed to
anchor calsequestrin to the Ca2+ release channel from the
lumenal side of the junctional face membrane (1-3).
Triadin was first detected as a 95-kDa integral membrane protein in
junctional SR vesicles isolated from skeletal muscle (4, 5).
Subsequently, its primary structure was deduced from the cDNA
sequence (6). The single transmembrane domain of skeletal muscle
triadin divides the protein into a short N-terminal cytoplasmic segment, and a highly charged C-terminal region, which accounts for the
bulk of the molecule, situated in the lumen of the SR (6-8). Guo and
Campbell (1) noticed an interaction of the lumenal domain of skeletal
muscle triadin with the Ca2+ release channel and
calsequestrin, which occurred in a
Ca2+-dependent manner. Others have suggested
that a region of the cytoplasmic domain can also interact with the
Ca2+ release channel depending on the Ca2+
concentration (9).
In cardiac muscle, the primary isoform of triadin expressed is triadin
1 (10), which accounts for greater than 95% of the total triadin
present in the heart. Triadin 1 (cardiac triadin) is a shorter variant
of skeletal muscle triadin, which appears to arise from alternative
splicing of mRNA transcribed from a common triadin gene (2, 10). On
SDS-PAGE, cardiac triadin runs as a doublet of 35- and 40-kDa molecular
mass proteins, corresponding to deglycosylated and glycosylated
mobility forms of the protein, respectively (10). Both cardiac and
skeletal muscle triadin are identical for the first 257 amino acids,
then cardiac triadin 1 ends abruptly, incorporating a short C-terminal
tail of 21 amino acids that is unique (2, 10). The regions common to
cardiac and skeletal muscle triadin are the short cytoplasmic segments (residues 2-47), the transmembrane segments (residues 48-68), and the
highly charged lumenal domains (residues 69-257). Notably, the highly
charged lumenal domains, which are common to all triadins, are
particularly enriched in repeating lysine and glutamic acid residues,
organized into "KEKE motifs," which have been proposed to
facilitate protein-protein interactions (11-13). It is not surprising, then, that, like skeletal muscle triadin, the lumenal domain of cardiac
triadin has also been shown to bind to both calsequestrin (2, 3,
14-16) and the ryanodine receptor (2, 3). Based on results of
immunoprecipation and fusion-protein pull-down assays, a quarternary
complex between triadin, junctin, calsequestrin, and the ryanodine
receptor was proposed, which may be important for the orchestrated
operation of Ca2+ release in both cardiac and skeletal
muscle (3). Supporting this idea, the calsequestrin-binding domain of
cardiac triadin was recently localized to a select run of only 25 amino
acids (residues 200-224), which incorporates a single KEKE motif (15), suggesting that the protein-protein interactions occurring between triadin and other components of the SR junction are very specific and
highly organized.
What is the precise function of cardiac triadin? This question has been
difficult to address experimentally due to the relative inaccessibility
of putative "structural" junctional SR proteins like triadin,
junctin, and calsequestrin to both biochemical and electrophysiological
study. An anchoring function for triadin (and junctin) seems obvious,
due to the ability of triadin (and junctin) to bind to both the
ryanodine receptor and calsequestrin when studied in vitro,
in combination with the ultrastructural observation that calsequestrin
appears to be adhered to the lumenal face of the ryanodine receptor
when observed in intact muscle with use of electron microscopy (17).
Beyond simply anchoring calsequestrin to the ryanodine receptor,
triadin may affect the channel activity of the ryanodine receptor
directly. For example, application of skeletal muscle triadin to the
purified ryanodine receptor incorporated in planar lipid bilayers
inhibits the channel activity of the protein (18, 19). As part of a
program to better understand the function of junctional SR proteins in
myocardium, we initiated a strategy in which key junctional SR proteins
are overexpressed in transgenic mouse hearts. With use of this approach for overexpression of cardiac calsequestrin, we demonstrated a greater
than 10-fold increase in caffeine-induced Ca2+ release in
transgenic cardiomyocytes (17), providing the first physiological
evidence implicating cardiac calsequestrin as the storage protein for
the lumenal Ca2+ that is released during muscle
contraction, an idea that has long been hypothesized, but difficult to
demonstrate experimentally. Using the same transgenic mouse model, we
observed that with overexpression of cardiac calsequestrin, the
associated junctional SR proteins triadin, junctin, and the ryanodine
receptor were down-regulated, whereas the free SR proteins SERCA2a and
phospholamban were up-regulated (17), showing for the first time that
protein expression at the junctional SR of the heart can be regulated
independently from protein expression at the free SR.
Here, we describe physiological experiments with transgenic mice
overexpressing canine triadin 1 in myocardium (10). Similar to results
obtained with forced overexpression of calsequestrin (17), we observed
that, with forced overexpression of triadin 1, selective
down-regulation of other proteins of the junctional SR can occur,
independently from changes in expression levels of proteins of the free
SR. In addition, the contractile phenotype of hearts from triadin
1-overexpressing mice is altered. Hearts from triadin 1-overexpressing
mice exhibit hypertrophy, impaired relaxation, blunted contractility
with increased pressure loading, and frequency-dependent
changes in myocyte shortening. Our results suggest that triadin 1 plays
an active role in Ca2+ release in the heart, beyond its
previously proposed structural role of anchoring of calsequestrin to
the ryanodine receptor.
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EXPERIMENTAL PROCEDURES |
Experimental Animals--
Generation of the transgenic mouse
lineage overexpressing canine triadin 1 was performed as described
recently (10). Screening for transgene-positive mice was done by
polymerase chain reaction (20, 21). All experiments described here were
performed on female mice 16-18 weeks of age. Animals were handled and
maintained according to approved protocols of the animal welfare
committees of the University of Münster (Münster, Germany)
and Indiana University (Indianapolis, IN).
Morphological Studies--
For macroscopic and histological
examination of mouse hearts, single longitudinal cuts were made along
the length of the myocardium. Hearts were then weighed and immediately
fixed in 4% buffered formalin, dehydrated, and embedded in paraffin.
Longitudinal tissue sections of 5 µm thickness were obtained from the
right and left ventricular free wall, and sections were stained with
hematoxylin-eosin, and periodic acid-Schiff reagent. Myocyte size was
quantified morphometrically with a computer-assisted image analysis
system (KS 300, Zeiss, Germany). The image analyzer was interfaced to a
video camera connected to a Zeiss microscope. Finally, each field was
transformed into a digital binary image. The mean cardiomyocyte diameter was calculated by measuring 100 cells/heart in the region of
the cell nucleus using the two-point distance function of the analysis
system. For electron microscopy, small pieces of left ventricular heart
tissue were fixed by immersion with 2.5% glutaraldehyde in 0.1 M phosphate buffer. After fixation, the specimens were further fixed in buffered 1% osmium tetraoxide for 2 h,
dehydrated in graded ethanol series, and embedded in Epon. Ultrathin
sections were cut and stained with uranyl acetate and lead citrate. The sections were investigated under a Philips CM 10 transmission electron microscope.
Measurement of Blood Pressure and Heart Rate--
The systolic,
mean, and diastolic blood pressures were measured in unanesthetized
mice with the tail-cuff method (22) and recorded on a monitor. Heart
rate was computed from the amplitude of the pulse signal. The mice were
held in the chamber for 10 min before the measurements were started. 25 single measurements made over a period of 5 days were averaged for each mouse.
Northern Blot Analysis--
Total RNA from single mouse
ventricles was isolated as described by Chomczynski and Sacchi (23). 20 µg of total cardiac RNA was separated by electrophoresis, transferred
to nylon membranes, and hybridized with dog triadin and mouse atrial
natriuretic factor (ANF) cDNA probes. For the generation of
radioactively labeled cDNA probes by reverse transcriptase-PCR
standard protocols were employed, using the cDNA as template in the
presence of
-32P-labeled nucleotides. Primers used had
the following sequences: 5'-GATATCGAATTCACCATGACTGAGA (forward, dog
triadin 1) and 3'-TCACTGTATCTGCTTCTTGCCC (reverse, dog triadin 1), and
5'-CGTGCCCCGACCCACGCCAGCATGGGCTCC (forward, mouse ANF) and
3'-GGCTCCGAGGGCCAGCGAGCAGAGCCCTCA (reverse, mouse ANF). PCR products of
expected sizes were obtained for triadin 1 (852 bp) and for ANF (393 bp). The identity of PCR products was confirmed by cycle sequencing
using an ABI-PRISM-310 automated sequencer (Applied Biosystems,
Weiterstadt, Germany).
Preparation of Homogenates and Microsomal Membranes--
For
analysis of crude homogenates from individual hearts, single hearts
from wild-type or triadin 1-overexpressing mice were homogenized at
4 °C for 1 min in 1.2 ml of medium containing 20 mM MOPS
(pH 7.0) and 0.25 M sucrose, using an Omni homogenizer (Omni International). The concentration of calcium in the homogenates was determined using Arsenazo III calcium reagent as recommended by the
manufacturer (Sigma). Homogenate protein was stored frozen at
40 °C. For preparation of cardiac microsomes partially enriched in
sarcoplasmic reticulum, 11 control ventricles or 11 transgenic ventricles were pooled and homogenized at 4 °C for 90 s in 25 ml of medium containing 0.25 M sucrose, 30 mM
histidine (pH 7.4), using a Polytron PT-10 homogenizer (Kinematica,
Lucerne, Switzerland) at a setting of 20,000 rpm. Control and
transgenic homogenates were centrifuged at 1,500 × gmax for 10 min at 4 °C, and the supernatants were collected and centrifuged at 45,000 × gmax for 30 min. The pellets from the second
spin were resuspended in 20 ml of 0.6 M KCl, 30 mM histidine (pH 7.0) and recentrifuged at 64,000 × gmax for 30 min. The KCl-extracted microsomes
were resuspended in 0.25 M sucrose, 10 mM
histidine (pH 7.0) and stored frozen in small aliquots at
40 °C.
SDS-PAGE, Immunoblotting, and Antibodies--
Homogenates or
microsomal samples were solubilized at room temperature in 5% SDS
buffer containing 62.5 mM Tris/HCl (pH 6.8), 5% glycerol,
40 mM dithiothreitol, and a trace of bromphenol blue. For
immunoblot analysis of all proteins other than the ryanodine receptor,
16-50 µg of homogenate or membrane protein were separated on 8%
SDS-PAGE according to the method of Porzio and Pearson (24). For
immunoblotting of ryanodine receptors from homogenate samples, 200 µg
of protein were applied per gel lane and electrophoresis was in 5%
polyacrylamide. After transferring of proteins to nitrocellulose, the
blots were incubated with different antibodies. The amount of bound
protein was detected by 125I-labeled protein A and
quantified using a PhosphorImager (Bio-Rad). The following antibodies
were used for detection of SR proteins: for the ryanodine receptor,
mouse monoclonal antibody 1E9 (3) was used; for SERCA2a, mouse
monoclonal antibody 2A7-A1 was used (16); for calsequestrin,
affinity-purified rabbit antibody raised to canine cardiac
calsequestrin was used (25); for junctin, affinity-purified rabbit
antibody raised to the C-terminal 15 amino acids of mouse junctin was
used; and for phospholamban, mouse monoclonal antibody 2D12 was used
(26). Two different antibodies were used for detection of triadin.
Mouse monoclonal antibody 8G5 was raised to recombinant canine triadin
1 expressed and purified from Sf21 insect cells (10). This
antibody did not cross-react with mouse triadin 1. The TRN6 antibody
was raised in a rabbit to a peptide corresponding to residues 146-160
of mouse triadin and affinity-purified (10). This antibody
cross-reacted with canine triadin but may not recognize canine triadin
as strongly as mouse triadin, because within the peptide used to
produce the antibody (residues 146-160 of mouse triadin), there are
three amino acid differences between mouse and dog triadin. Protein concentrations were determined according to the method of Lowry et al. (27).
[3H]Ryanodine Binding Assay--
For detection of
[3H]ryanodine binding to ryanodine receptors, 400 µg of
homogenate protein were incubated at 37 °C for 60 min in 200 µl of
buffer containing 20 mM MOPS (pH 7.1), 1 mM
CaCl2, 0.6 M NaCl, and 21 nM
[3H]ryanodine. Nonspecific binding was determined in the
same buffer containing in addition 10 µM cold ryanodine.
Samples were filtered, rinsed repetitively with ice-cold saline, and
the remaining radioactivity on the filters was quantified in a liquid
scintillation counter (16).
Ca2+-ATPase Assay--
Ca2+-ATPase
activity of microsomal membranes was measured at 37 °C in buffer
containing 50 mM MOPS (pH 7.2), 3 mM
MgCl2, 100 mM KCl, 5 mM
NaN3, 3 µg/ml A23187, 1 mM EGTA, and 0-1.0
mM added CaCl2 to give the desired ionized
Ca2+ concentrations (28). Pi release from ATP
was measured colorimetrically, and
Ca2+-dependent activities were analyzed.
Isolation of Cardiomyocytes--
Ventricular myocytes were
isolated from control and triadin 1-overexpressing mouse hearts using a
published protocol (29). Animals were pretreated with heparin (5 units/g body weight), and later anesthetized with CO2.
Mouse hearts were excised, and the cannulated aorta was fixed to a
Langendorff apparatus. Hearts were perfused for 5 min at 2 ml/min with
a Ca2+-free solution (solution A) composed of (in
mM) 140 NaCl, 5.8 KCl, 0.5 KH2PO4,
0.4 NaH2PO4, 0.9 MgSO4, 10 HEPES,
11.1 glucose (pH 7.1), followed by a perfusion for 30 min with solution
A supplemented with 0.2 mg/ml collagenase (type D, Roche Molecular
Biochemicals). Ca2+ concentration was gradually increased
during digestion to 100 µM. After enzymatic digestion,
the hearts were perfused for 10 min with solution A. The ventricles
were cut into several pieces and subjected to gentle agitation through
a nylon mesh to separate the myocytes.
Measurement of Ca2+ Transient
[Ca]i--
Cardiomyocytes were incubated for 5 min at
room temperature with solution A containing in addition 2.5 mM CaCl2, 50 mg/l ascorbic acid (solution B),
and 25 µM Indo-1/AM. The cells were then superfused with
solution B without dye for 45 min before the measurements started.
Indo-1 fluorescence was recorded at room temperature from single
myocytes using a dual-emission microfluorescence system (Photon
Technologies Inc., South Brunswick, NJ). Excitation was at 365 nm, and
the emitted fluorescence was recorded at 405 and 495 nm. The ratio of
fluorescence at the two wavelengths was used as an index of cytosolic
Ca2+ concentration. Cardiomyocytes were stimulated at 0.5 Hz with platinum electrodes placed on the sides of the experimental
chamber. Data were collected at 20 Hz, and acquisition and processing
were supported by Felix 1.1 software (Photon Technologies Inc., South Brunswick, NJ).
Cardiomyocyte Shortening Measurements--
Contractility of
isolated cardiomyocytes was recorded with a Panasonic video recorder
AG-7350 (Matsushita Electric Ind. Co., Japan). Cardiomyocyte shortening
was visualized on a monitor (Pieper WV-5410, Pieper AG, Mellingen,
Switzerland), and connected to a video integrator (Pieper Video
Integrator 310A) interfaced to a video camera (Pieper Kamera
FK7512-IQ). The video camera was attached to a Leica microscope
(Wetzlar, Germany). Edge detection measurements were performed at room
temperature on intact, rod-shaped cells, which had no spontaneous
contractions or microblebs. Cells were placed in a chamber (1 ml) and
perfused at 0.5-1 ml/min with solution A containing 1 mM
CaCl2. Cardiomyocytes were electrically stimulated at the
indicated frequencies. The maximal magnitude of contraction was
normalized to resting cell length and expressed as percentage of shortening.
Whole-cell L-type Ca2+ Current
(ICa)--
Single cardiomyocytes were studied using the
whole-cell variation of the patch-clamp technique. Recordings were
performed under conditions that suppress Na+ and
K+ currents (30). Briefly, cells were plated in a small
dish (2 ml) on the stage of an inverted microscope (Leica). The
extracellular solution was composed of (in mM): TEA-Cl 130, MgCl2 1, 4-aminopyridine 4, HEPES 10, dextrose 10, CaCl2 2, adjusted to pH 7.3 with TEA-OH. The micropipette
electrodes (resistances 1.5-2.5 megohms) were filled with (in
mM): potassium aspartate 80, KCl 50, KH2PO4 10, MgCl2 0.5, MgATP 3, HEPES 10, EGTA 1, adjusted to pH 7.4 with KOH. All experiments were
done at room temperature. Unless stated otherwise, currents were
elicited by voltage steps from a holding potential of
40 mV to a test
potential of +10 mV for 200 ms, applied every 10 s. Cell
capacitance and ICa were recorded with an L/M-PC
amplifier (LIST-Electronic, Darmstadt, Germany) according to
standard protocols. Data were computed with the ISO2 software (MFK,
Niedernhausen, Germany).
Measurement of Cardiac Contractile Parameters in Work-performing
Heart Preparations--
Work-performing heart preparations were
utilized as described previously (31). Mice were anesthetized
intraperitoneally with 2.0 g/kg body weight urethane and treated with
1.5 units of heparin. Hearts were removed from the opened chest,
immediately attached by the aorta to a 20-gauge cannula, and perfused
retrogradely with oxygenized Krebs-Henseleit buffer (37.4 °C)
containing (mM) NaCl 118, NaHCO3 25, Na-EDTA 0.5, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, and glucose 11. During the short period of
retrograde perfusion, the pulmonary vein was cannulated. The perfusion
of the heart was then changed to an anterograde mode. The venous return
(preload) and the aortic pressure (afterload) were adjusted to 5 ml/min
cardiac output and 50 mmHg, respectively. Loading conditions were
changed then by increasing the aortic pressure (afterload) to 60 mmHg,
while keeping the venous return constant. Heart rate, aortic pressure,
left intraventricular pressure (systolic, diastolic, and end
diastolic), and atrial pressure were measured and monitored
continuously. The first derivative of left intraventricular pressure
(+dP/dt and
dP/dt), time
to peak pressure/mmHg, and time to half-relaxation/mmHg were calculated
with a computer program (BeMon 2.1, Ingenieurbüro Jäckel,
Hanau, Germany). Cardiac output and coronary flow were calculated from
the parameters above.
Materials--
[3H]Ryanodine,
[
-32P]-dCTP, and 125I-labeled protein A
were obtained from Perkin Elmer Life Sciences. Ryanodine was
purchased from S. B. Penick. Indo-1/AM was supplied by Sigma. All
other chemicals were of reagent grade.
Statistical Analysis--
Data are reported as means ± S.E. Statistical differences between the different types of mice were
calculated by analysis of variance followed by Bonferroni's
t test. p < 0.05 was considered significant.
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RESULTS |
Triadin 1-overexpressing Mice--
We used the
-myosin heavy
chain promoter to drive cardiac-specific overexpression of canine
triadin 1 in mouse hearts (32). Northern blot analysis using a probe
specific for canine triadin 1 revealed abundant mRNA production by
the transgene (Fig. 1A). Selective overexpression of canine triadin 1 in transgenic mouse hearts
was demonstrated by immunoblotting cardiac microsomes with the triadin
monoclonal antibody 8G5-G3, which detects only the canine isoform of
the protein (Fig. 1B). The two prominent mobility forms of
triadin 1 (T1) detected, of molecular masses 35 and 40 kDa, were
reported previously and correspond to deglycosylated and glycosylated
(
) forms of the protein, respectively (10). When the same membrane
samples were probed with the TRN6 antibody, which recognizes both mouse
and dog triadin 1, the substantial overexpression of triadin 1 in
transgenic membranes was evident (Fig. 1C). The level of the
triadin 1 (T1) signal was increased by 5-fold (Table II) in microsomes
from triadin 1-overexpressing mice compared with microsomes from
wild-type mice, when using the TRN6 antibody to estimate protein
overexpression. The TRN6 antibody may underestimate overexpression of
canine triadin 1 somewhat, however, because this antibody was raised to
amino acid residues 146-160 of mouse triadin, which are only 80%
conserved in dog triadin. Overexpressed triadin 1 was easily visible on SDS-PAGE and Coomassie Blue staining of cardiac microsomes from transgenic mice, but endogenous triadin 1 could not be visualized with
certainty when using microsomes from control mice (data not shown).

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Fig. 1.
Identification of triadin 1. A, expression of triadin mRNA in transgenic hearts.
Total RNA (20 µg) from WT and triadin 1-overexpressing
(TRD) mouse hearts was hybridized with a
32P-labeled dog triadin 1-specific probe. B and
C, immunodetection of triadin 1. 35 µg of protein of
ventricular microsomes from wild-type and triadin 1-overexpressing mice
were electrophoresed, transferred to nitrocellulose, and probed with
mouse monoclonal antibody 8G5 raised to canine triadin 1 (dog TRD) or
rabbit antibodies raised to residues 140-160 of mouse triadin 1 (mouse/dog TRD). The 35- and 40-kDa molecular mass forms of triadin 1 (T1) are bracketed. denotes the glycosylated
form of T1 with an apparent molecular mass of 40 kDa.
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Cardiac Hypertrophy--
Triadin 1-overexpressing hearts from
adult mice (16-18 weeks of age) exhibited a mild degree of
hypertrophy, as indicated by an increase in heart mass and
heart to body weight ratio relative to
hearts from wild-type mice (Fig. 2A, Table
I). We observed an enhanced heart weight
and heart to body weight ratio already in 10-week-old triadin
1-overexpressing mice. However, all triadin 1-overexpressing mice
studied (10-18 months of age) had a normal mortality compared with
control mice. Moreover, in contrast to results recently obtained with
calsequestrin-overexpressing mice (17, 33), triadin 1-overexpressing
mice showed no overt signs of heart failure (e.g. no ascites
or pulmonary congestion). Interestingly, triadin 1-overexpressing
hearts were covered with white fibrotic plaques of varying size and
shape (Fig. 2A). We believe these plaques contained large
amounts of Ca2+, because the cardiac homogenates from
triadin 1-overexpressing mice contained substantially more
Ca2+ than did the homogenates from control mice (2.84 ± 0.66 mM [Ca2+] versus 0.61 ± 0.07 mM [Ca2+], in homogenates from
triadin 1-overexpressing and control mouse hearts, respectively;
n = 7; p < 0.05). Ca2+
carry-over from triadin 1-overexpressing heart homogenates was sufficient to interfere significantly with Ca2+ response
assays on [3H]ryanodine binding and
Ca2+-ATPase activities of crude homogenates. However, we
observed that the material containing the contaminating
Ca2+ could be removed by low speed centrifugation at
1,500 × gmax. Consequently, the
Ca2+ response experiments were conducted with cardiac
microsomes, which were free from the interfering Ca2+.

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Fig. 2.
Overexpression of triadin 1 results in
cardiac hypertrophy. A, representative hearts
from WT and triadin 1-overexpressing (TRD) mice. The
transgenic left atrium (LA) and ventricle (LV)
are enlarged as indicated by arrows, and white fibrous
material is adhered to the surface of the transgenic ventricle.
B, ultrastructural electron microscope analysis of
cardiomyocytes in control and triadin 1-overexpressing hearts. Note
that the myofibrils are disrupted. Magnification, ×13,500.
RA, right atrium; RV, right ventricle.
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Table I
Triadin overexpression is accompanied by cardiac hypertrophy
Body weight and heart weight were determined for WT and triadin
1-overexpressing (TRD) mice. The cardiomyocyte diameter was measured on
hematoxylin-eosin and periodic acid-Schiff-stained longitudinal cuts of
the left ventricle, as described under "Experimental Procedures."
The indicated values are mean ± S.E. *,
p < 0.05 vs. WT.
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Longitudinal sections showed an increase of wall thickness in both
ventricular chambers from triadin 1-overexpressing mice, and
histological examination revealed that triadin 1-overexpressing cardiomyocytes from right and left ventricles were bigger than control
cardiomyocytes (Table I). Electron microscope analysis (using
16-18-week-old mice) revealed that myofibrillar structure was
disrupted with partial separations occurring in single fibers (Fig.
2B). In addition, the myofibrils were frequently displaced by an undefined electron-dense matrix and the mitochondria were disorganized. The myofibrillar abnormalities noted along with the
mitochondrial disorganization are indicators of a diseased heart in
early stages of failure, although no overt failure was noted in the
animals. In sharp contrast to calsequestrin-overexpressing mice (17),
the microarchitecture of the junctions between the SR and the surface
membrane or between the SR and T-tubules was not obviously disturbed.
Hypertrophy was also accompanied by a 4-fold higher ANF mRNA
expression in triadin 1-overexpressing mouse hearts. Mean blood
pressure and heart rate were comparable between triadin
1-overexpressing mice and wild-type mice (data not shown).
Expression Levels of Other SR Proteins--
To see if
overexpression of triadin 1 in transgenic mouse hearts altered the
expression levels of other SR proteins, we immunoblotted crude cardiac
homogenates from control and triadin 1-overexpressing mice, using an
array of antibodies raised to different SR
proteins. Proteins arising from free or
junctional SR membranes were analyzed (Fig. 3, Table
II). Expression levels of SERCA2a and
phospholamban, proteins originating from free SR (16, 28), were
unaltered in triadin 1-overexpressing homogenates. Likewise, the
expression level of calsequestrin, the intralumenal
Ca2+-binding protein localized to junctional SR (17, 28),
was unchanged. In contrast, the expression levels of the two other junctional SR proteins analyzed, the ryanodine receptor and junctin (17, 28), were decreased by 55% and 73%, respectively, when assessed
by immunoblotting. The structural and sequence similarities between
triadin and junctin were noted previously (10, 16), and it is
interesting that junctin was the SR protein most down-regulated in
heart, when triadin 1 was overexpressed. The down-regulation of the
ryanodine receptor in triadin 1-overexpressing homogenates detected by
immunoblot assay was confirmed by [3H]ryanodine binding
assay, in which the content of [3H]ryanodine binding
sites in transgenic homogenates was decreased by 30% (Table II).

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Fig. 3.
SR protein levels in homogenates from
transgenic hearts. Control (C) and triadin
1-overexpressing (T) heart homogenates were subjected to
immunoblot analyses. Identical amounts of protein were loaded per lane
for each antibody utilized. Blots were probed with antibodies specific
for ryanodine receptor (RyR), Ca2+ pump
(SERCA2a), calsequestrin (CSQ), triadin 1 (TRD), junctin (JCN), and phospholamban
(PLB) as described under "Experimental
Procedures".
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Table II
Levels of SR proteins in homogenates from wild-type and triadin
1-overexpressing mouse hearts
Levels of cardiac regulatory SR proteins in homogenates of WT and
triadin 1-overexpressing (TRD) mouse hearts were determined after
scanning the 125I-labeled immunoblots in a GS-250 Molecular
Imager (Bio-Rad). Note that phosphorimager values are not directly
comparable between different proteins analyzed, because different
antibodies and different phosphorimager exposure times were used for
detection of each protein. Results were obtained from the immunoblot
depicted in Fig. 3 (n = 7). [3H]Ryanodine
binding was also measured in homogenates obtained from 12 sets of
control and transgenic littermates, including those sets used for the
immunoblot analysis. Values are means ± S.E. (n = hearts). *, p < 0.05 vs. WT.
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Ca2+ Concentration Effects on
[3H]Ryanodine Binding and Ca2+-ATPase
Activity of Cardiac Microsomes--
To test if triadin 1 overexpression in transgenic mouse hearts had an effect on
Ca2+ activation of opening of the Ca2+ release
channel or of Ca2+ activation of ATP hydrolysis by SERCA2a,
we measured [3H]ryanodine binding and
Ca2+-ATPase activity in cardiac microsomes prepared from
control and triadin 1-overexpressing mouse hearts.
[3H]Ryanodine binding assay was used as an indirect
assessment of the open state of the Ca2+ release channel
(34). Ca2+ increased [3H]ryanodine binding
over a similar range of Ca2+ concentrations for wild-type
and triadin 1-overexpressing membranes, suggesting that triadin 1 overexpression did not alter the ability of the ryanodine receptor to
sense Ca2+ ions under the buffer conditions utilized.
Half-maximal activation of [3H]ryanodine binding occurred
at 0.402 and 0.475 µM Ca2+ for control and
transgenic membranes, respectively. Likewise, the Ca2+
concentration required for activation of ATP hydrolysis by the Ca2+ pump was not significantly altered by triadin 1 overexpression in SR vesicles isolated from triadin 1-overexpressing
mouse hearts (data not shown).
Ca2+ Transients [Ca]iand Cell
Shortening--
To test for alterations of intracellular
Ca2+ handling in triadin 1-overexpressing hearts, we
measured resting cytoplasmic Ca2+concentration,
Ca2+ transients, and contractility in isolated ventricular
myocytes from wild-type and transgenic mice. Myocytes were loaded with Indo-1 and stimulated at 0.5 Hz. Diastolic Ca2+ levels were
marginally but significantly elevated in cells from triadin
1-overexpressing hearts compared with wild-type cells (Table
III). The maximal amplitude of the
Ca2+ transient was comparable between triadin
1-overexpressing and wild-type cardiomyocytes (Table III); however, the
rate of decay of the Ca2+ transient was markedly decreased
in triadin 1-overexpressing cardiomyocytes. The time to 50% decay of
the Ca2+ transient was increased by 74%. To determine how
the altered [Ca]i kinetics influenced
contractility, we measured cardiomyocyte shortening using an edge
detection system. Maximal cell shortening was depressed by 72% in
triadin 1-overexpressing cardiomyocytes (Table III), and, consistent
with Ca2+ transient observations, the kinetics of
shortening were prolonged, with transgenic myocytes taking a 23%
longer time to relax than control myocytes (Table III).
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Table III
Ca2+ transients and contractile parameters of isolated
cardiomyocytes
Indo-1 signals were determined in isolated ventricular cardiomyocytes
from WT and triadin 1-overexpressing (TRD) mice. The contractile
measurements were performed by edge detection. Isolated myocytes were
paced at 0.5 Hz. Lmax indicates the maximal myocyte
length, and Lmin the minimum myocyte length. Values
represent the mean ± S.E. (n = cardiomyocytes).
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Cardiomyocyte Shortening-frequency Relationship--
A correlation
between an impaired SR-Ca2+ handling and an altered
force-frequency relationship in myocardium has been described previously (35, 36). Therefore, we compared the shortening-frequency relationship in isolated cardiomyocytes from wild-type and triadin 1-overexpressing mice. Remarkably, the contractile abnormalities noted
above observed at low stimulation frequencies between 0.5 to 3 Hz
disappeared when the stimulation frequency was increased to 4-5 Hz
(Fig. 4). These results demonstrate that
the depressant effect of triadin 1 overexpression on myocardial
contractility is rate-dependent, with the major effects
occurring at very low stimulation frequencies which are not typically
encountered in normally beating mouse hearts.

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Fig. 4.
Shortening-frequency relationship of
wild-type and triadin 1-overexpressing cardiomyocytes. The
frequency-dependent shortening was determined in WT and
triadin 1-overexpressing (TRD) cardiomyocytes. Stimulation
of cells was started at 0.5 Hz and stepwise increased up to 5 Hz at
5-min intervals (abscissa). Ordinate is given as
the percentage of systolic shortening. Data are mean ± S.E.
(n = cardiomyocytes).
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Contractile Parameters in Work-performing Heart
Preparations--
To see if contractile abnormalities in triadin
1-overexpressing cardiomyocytes persisted to the whole heart level,
isolated hearts from wild-type and triadin 1-overexpressing mice were
perfused in the work-performing mode. Under basal loading conditions
utilized (50 mmHg aortic pressure), triadin 1-overexpressing mice and
wild-type mice exhibited similar spontaneous heart rates and similar
levels of systolic intraventricular pressure (Table
IV). However, the maximal rate of
relaxation (
dP/dt) was decreased by 18% in
triadin 1-overexpressing hearts, and relaxation times were prolonged
(Table IV). Triadin 1-overexpressing hearts responded to increased
afterload with blunted contractility. The rates of contraction
(+dP/dt) and rates of relaxation
(
dP/dt) were decreased at 60 mmHg by 15% and
20%, respectively (Table IV). Furthermore, the systolic and diastolic
intraventricular pressures were lower in triadin 1-overexpressing
hearts (Table IV).
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Table IV
Work-performing heart preparations
The contractile parameters of WT and triadin 1-overexpressing (TRD)
mouse hearts (16-18 weeks of age) were studied at an afterload of 50 and 60 mmHg using the work-performing mode. LV indicates left
ventricle, +dP/dt is the maximal rate of left
ventricular pressure development, and dP/dt is
the maximal rate of left ventricular pressure decline. TPP is the time
to peak pressure/mmHg, RT1/2 is the time to
half-relaxation/mmHg, and t50 is the time to 50%
relaxation. The indicated values are mean ± S.E.
(n = 5). *, p < 0.05 vs. WT 50 mmHg; **, p < 0.05 vs. WT 60 mmHg.
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Characteristics of L-type Ca2+ Channel--
To test
whether the altered Ca2+ handling in triadin
1-overexpressing cardiomyocytes was accompanied by changes in the
trigger for SR-Ca2+ release, we measured the
L-type Ca2+ channel current
(ICa) in control and triadin 1-overexpressing cardiomyocytes. Cell membrane capacitance was increased in triadin 1-overexpressing cardiomyocytes compared with wild-type myocytes (241.4 ± 9.3 pA versus 196.5 ± 7.9 pA,
respectively; n = 10-14; p < 0.05),
consistent with greater size of the transgenic cardiomyocytes observed
in the histological examination. The kinetics of inactivation of the
L-type Ca2+ channel was impaired in triadin
1-overexpressing cells. The fast time constant of inactivation of
ICa,
1, was increased by 20% in
triadin 1-overexpressing cardiomyocytes (6.5 ± 0.4 ms in
transgenic versus 5.4 ± 0.2 ms in control,
n = 10-14, p < 0.05), whereas the
slow time constant of inactivation of ICa,
2, was not significantly altered (41.6 ± 1.8 ms in
transgenic versus 46.5 ± 2.8 ms in control, n = 10-14). At depolarizing potentials between +20 to
+30 mV, the normalized ICa current densities
were reduced in triadin 1-overexpressing cardiomyocytes (Fig.
5).

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Fig. 5.
ICa in wild-type and
triadin 1-overexpressing cardiomyocytes. The peak current-voltage
relationships obtained from WT and triadin 1-overexpressing
(TRD) cardiomyocytes. Ca2+ channels were
activated by 200-ms depolarizing pulses from a holding potential of
40 mV to the indicated test potentials. Abscissa, voltage
in mV. Ordinates are given in currents divided by the cell
capacitances (pA/pF). Data are mean ± S.E. (n = cardiomyocytes).
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DISCUSSION |
Cardiac triadin is localized in the junctional SR, where it
appears to "anchor" calsequestrin to the Ca2+ release
channel. The functional consequences of this interaction in the heart
have not previously been investigated. Here, we used a transgenic model
of triadin 1 overexpression in the heart to show that overexpression of
triadin 1 produces selective down-regulation of other junctional SR
proteins, cardiac hypertrophy, and contractile abnormalities, with the
most notable effect being on cardiac relaxation parameters. In
addition, our study suggests that the triadin 1 protein level is an
important determinant of the force-frequency relationship in myocardium.
What is the reason for the hypertrophy observed in triadin
1-overexpressing mice? Compared with the hypertrophy observed with overexpression of the associated junctional SR protein, calsequestrin, which gives striking cardiomegaly (2-fold increase in heart weight) and
progresses rapidly to heart failure and early death (17), the
hypertrophy associated with triadin 1 overexpression is relatively benign (16% increase in heart to body weight ratio at 16-18 weeks). It does not appear to yield a phenotype of overt heart failure or early
death. Nonetheless, key indicators of hypertrophy and failure, like
elevated ANF and increased myocyte size and increased cell capacitance,
are evident in triadin 1-overexpressing mice. Cardiac hypertrophy is a
fundamental adaptation of the adult heart to mechanical stress and
humoral factors but can also occur from primary alterations in cardiac
gene expression (37). The final cause is an increased protein
synthesis, possibly combined with a prolonged half-life of cardiac
structural proteins. Postnatal cardiomyocytes are not capable of cell
division, but they can adapt to load by an increase of cardiomyocyte
size, changes in the rate of protein synthesis, and reactivation of a
fetal gene regulatory program (38-40). In recent studies, it has been
shown that an increased diastolic intracellular Ca2+ level
can act as a hypertrophic signal (41-43). However, diastolic Ca2+ was only slightly increased in the present
overexpression model. Although the maximal amplitude of the
Ca2+ transient was not changed in triadin 1-overexpressing
cardiomyocytes, the Ca2+ transient was prolonged,
suggesting that the average intracellular Ca2+ may be
increased. Unexplicably, however, we observed a significant increase in
plaque-like material on the surfaces of triadin 1-overexpressing mouse
hearts, which appeared to be composed partially of Ca2+
concretions. The total Ca2+ content of triadin
1-overexpressing heart homogenates was substantially increased, but the
origin of "extra" Ca2+ remains undefined at present.
Recent findings by Carrion and co-workers (44) demonstrated that even
slight elevations of Ca2+ can stimulate the DNA-binding
transcriptional regulator protein DREAM, thus inducing the expression
of several growth genes. This pathway was considered in a transgenic
mouse model with targeted overexpression of the
1
subunit of the L-type Ca2+ channel, which
exhibited a mild cardiac hypertrophy (45). At present, however, we can
only speculate which Ca2+-activated pathway may lead to the
hypertrophy observed in the present model.
Interestingly, transgenic hearts adapted to overexpression of triadin 1 by down-regulating the levels of two other junctional proteins involved
in Ca2+ release function, the Ca2+ release
channel and junctin. The level of the other junctional SR protein,
calsequestrin, remained unchanged, as did the levels of the two free SR
proteins, the Ca2+ pump and its associated regulatory
protein phospholamban. It may be that, in attempting to hold the amount
of Ca2+ release from the junctional SR as close to normal
as possible when triadin 1 is overexpressed, the SR adapts by
down-regulating other junctional proteins, including the ryanodine
receptor and junctin. Calsequestrin may remain unchanged acting as a
"set point" or "barometer" for maintaining intralumenal
Ca2+ constant, whereas the other junctional SR proteins may
adapt around this set point to maintain the net Ca2+
release as close to normal as possible. The protein most down-regulated with triadin 1 overexpression, junctin, is the protein most homologous to triadin, providing additional evidence that the two proteins have
similar functions, i.e. anchoring calsequestrin to the
ryanodine receptor and modulating Ca2+ release (3, 15).
Interestingly, immunoblot analysis indicated that the protein level of
the ryanodine receptor was down-regulated 55% in triadin
1-overexpressing mouse hearts, whereas the [3H]ryanodine
binding assay suggested that the ryanodine receptor was down-regulated
only 30%. Since ryanodine only binds to open Ca2+ release
channels, it could be that in triadin 1-overexpressing hearts,
compensatory biochemical changes are occurring allowing the ryanodine
receptor to become more easily activable, thus partially offsetting the
substantial decrease in the total level of receptors that are present.
However, the ryanodine receptor in triadin 1-overexpressing mouse heart
microsomes was no more responsive to Ca2+ than was the
ryanodine receptor in control heart microsomes. It is possible that
other regulatory factors are involved.
The major physiological effects of triadin 1 overexpression on
Ca2+ transients and contractility of cardiac myocytes
occurred at low stimulation frequencies and were predominately on
relaxation indices. For example, decay of the Ca2+
transient, rate of cell shortening, and rate of whole heart relaxation (at basal loading conditions) were substantially prolonged in triadin
1-overexpressing myocytes. The maximal extent of cell shortening was
also substantially reduced, however. These observations suggest that,
at low stimulation frequencies, the net reuptake rate of
Ca2+ by the SR, as well as the net release of
Ca2+, may be substantially impaired. The biochemical cause
(or causes) for these physiological changes are difficult to ascribe
directly to over- or underexpression of a single protein, because so
many proteins are changing concurrently. We can conclude that triadin 1 overexpression in the heart has significant physiological effects on
cardiac contractility, which includes aberrations in both net Ca2+ uptake and Ca2+ release by the SR. Other
effects, such as prolongation of ICa current
kinetics, are probably secondary to impaired Ca2+ release
from the SR and not reflective of a direct interaction between triadin
1 and proteins of the sarcolemma such as the L-type Ca2+ channel. The impaired contractility detected occurred
mostly at low stimulation frequencies in triadin 1-overexpressing mice, whereas at higher stimulation frequencies, which are closer to normal
for the mouse, the contractile responses were similar.
These frequency-dependent contractile changes may imply
that the altered junctional SR protein levels, including profound down-regulation of junctin, are adaptive responses occurring to maintain SR Ca2+ release/homeostasis as close to normal as
possible at the normal mouse heart rate. It is conceivable that at
lower heart rates the protein adaptations are no longer appropriate and
contractility regulation breaks down. As a result, the SR can no longer
regulate Ca2+ with fidelity, and contractile changes become
obvious (Fig. 4).
In summary, we have shown that triadin 1 plays an important role in the
force-frequency relationship. It may be that adult triadin
1-overexpressing mice escape overt heart failure because other
junctional SR proteins down-regulate to allow Ca2+ release
to remain close to normal at the normal mouse heart rate. To test this
hypothesis, we are currently mating junctin-overexpressing mice (46)
with triadin 1-overexpressing mice so that forced overexpression of
both proteins can occur simultaneously.