From the Departments of Physiology and
¶ Surgery, School of Medicine, University of Michigan, Ann
Arbor, Michigan 48109-0622
Received for publication, December 10, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Thin filament proteins tropomyosin (Tm), troponin
T (TnT), and troponin I (TnI) form an allosteric regulatory complex
that is required for normal cardiac contraction. Multiple isoforms of
TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and
40-50% of native Tm or TnT were specifically replaced. The
Ca2+ sensitivity of tension development was markedly
enhanced by TnI replacement but not by TnT or Tm isoform replacement.
Titration of TnI replacement from >90% to <30% revealed a dominant
functional effect of slow skeletal TnI to modulate regulation.
Over this range of isoform replacement, TnI, but not Tm or TnT
embryonic isoforms, influenced calcium regulation of contraction, and
this identifies TnI as a potential target to modify contractile
performance in normal and diseased myocardium.
Sarcomeric thin filament proteins form an allosteric regulatory
complex that governs Ca2+-activated contraction in the
myocardium (1-3). Prior to contraction, intracellular Ca2+
levels are low and the thin filament regulatory proteins block strong
actin-myosin interactions to inhibit force generation. Upon release of
Ca2+ from intracellular stores to initiate contraction,
Ca2+ binding to troponin C (TnC) causes conformational
changes within the regulatory apparatus: tropomyosin
(Tm),1 troponin I (TnI), and
troponin T (TnT). Consequently, strong interactions between actin and
myosin are permitted and force is generated. This precise interplay
between Ca2+ and the thin filament regulatory complex is
essential for orchestrating normal beat-to-beat regulation of
cardiac performance.
The isoform content of the thin filament regulatory complex changes
during cardiac development (Fig. 1) and in numerous cardiac disease
states. Specifically, The structural complexities of the multimeric thin filament assembly
and the overlapping pattern of TnI, Tm and TnT isoform switching in
cardiac development have made it difficult to assign specific
functional outcomes to each regulatory isoform transition (4). The
cellular consequences of disease-mediated disregulation in TnT and Tm
isoform expression in cardiac myocytes also remain unknown. Cardiac
transgenic studies have proved to be extremely valuable in
understanding organ and organismal outcomes to modifications in thin
filament regulatory isoforms (17, 18). One potential consideration in
using this approach lies in distinguishing, at the cellular level, the
primary effects of transgene expression from possible
secondary/compensatory effects, including the cardiac histopathology
and maladaptive growth that can arise in transgenic mice (19, 20). In
addition, to date there has been no single study comparing the
functional significance of a systematic manipulation of
cardiac-expressed Tm, TnT, and TnI thin filament regulatory isoforms in
adult cardiac myocytes.
We tested the hypothesis that systematic replacement of each embryonic
isoform would uniquely modify adult myocyte contractile responses to
Ca2+. To this end, recombinant viral vectors were used for
acute genetic engineering of the thin filament regulatory complex. This
system has been demonstrated to cause stoichiometric replacement of
native thin filament regulatory proteins (TnI, TnT, or Tm) (21-29).
Self-protein (e.g. expression of adult isoforms, cTnI, aTnT,
Recombinant Adenoviral Vectors--
Eight recombinant adenoviral
vectors were used in this study: cTnI; cTnIFLAG (C-terminal FLAG
epitope-tagged cTnI); ssTnI, using rat cDNAs; Adult Cardiac Myocyte Isolation, Primary Culture, and Gene
Transfer--
These methods have been previously described in detail
(31). Briefly, hearts were removed from adult female Sprague-Dawley rats, mounted on a modified Langendorff perfusion apparatus, perfused with Krebs-Henseleit buffer (KHB) (in mM/liter, 118 NaCl,
4.8 KCl, 25 HEPES, 1.25 K2HPO4, 1.25 MgSO4, 11 glucose) containing 1 mM
Ca2+ for 5 min, and then perfused with KHB lacking added
Ca2+. After 5 min, collagenase (0.5 mg/ml) and
hyaluronidase (0.2 mg/ml) were added to the Ca2+-free KHB
perfusate, and after 15 min of perfusion the Ca2+
concentration was gradually increased to 1 mM and perfused
for an additional 15 min. The heart was removed to a sterile beaker, and the ventricles were gently minced in enzyme solution (1 mM Ca2+-KHB + 0.5 mg/ml collagenase + 0.2 mg/ml
hyaluronidase). Minced tissue was gently swirled in a 37 °C water
bath for 4 × 5 min. The final 2 digests were collected,
centrifuged at 45 × g for 10 s, and pelleted
cells were resuspended in 1 mM Ca2+-KHB + 20 mg/ml bovine serum albumin before gradually increasing the solution
Ca2+ to 1.75 mM. Cells were then resuspended in
Dulbecco's modified Eagle's medium + 5% fetal bovine serum + penicillin/streptomycin, counted, and plated on
18-mm2 laminin-coated glass coverslips at 2 × 104 myocytes/coverslip for 2 h. For cardiac myocyte
gene transfer, medium was gently replaced with 200 µl of
adenovirus diluted in Dulbecco's modified Eagle's medium + P/S to the
desired titer. The multiplicity of infection ranged from
250-500 plaque forming units (pfu/rod-shaped myocyte) to optimize gene
transfer efficiency for each vector tested. One hour later, 2 ml of
Dulbecco's modified Eagle's medium + P/S was added to each coverslip.
Medium was replaced with fresh Dulbecco's modified Eagle's medium + P/S 24 h later and every 2-3 days thereafter for up to 7 days
post-gene transfer.
Immunoblotting--
Glass micropipettes were used to collect
cardiac myocytes for subsequent gel analysis. The non-ionic detergent
Triton X-100 (TX-100) was used to permeabilize the sarcolemma for
comparison to membrane-intact myocytes prior to loading. SDS-PAGE was
run at a constant current of 20 mA for 4-5 h, then transblotted onto polyvinylidene difluoride membrane (0.45 µm, Millipore) for 2000 volt-hours and fixed in PBS containing 0.25% glutaraldehyde. Western blots were carried out by initially blocking nonspecific binding sites
with Tris-buffered saline containing 5% nonfat dry milk for 2 h.
Blots were then incubated with either an anti-cardiac TnT monoclonal
antibody (Chemicon mAb 1695, 1:500), an anti-striated muscle TnT
monoclonal antibody (Sigma T-6277, clone JLT-12, 1:200), an anti-Tm
monoclonal antibody (Sigma T-2780, clone TM311, 1:1 × 106), or an anti-TnI antibody (Chemicon mAb 1691, 1:500)
for 2 h. To further validate isoform expression, anti-TnI antibody
M32259 (Fitzgerald Industries International, Inc, Concord, MA) and
anti-Tm antibodies CH-1 were used. Primary antibody was detected by
peroxidase-conjugated goat anti-mouse IgG (1:1000) and an enhanced
chemiluminescence detection assay (Amersham Biosciences).
Indirect Immunofluorescence--
Myocytes on coverslips were
fixed in 3% paraformaldehyde, washed with PBS, and treated with 50 mM ammonium chloride to remove excess aldehydes.
Nonspecific binding of antibodies was minimized with blocking solution
containing PBS with 20% normal goat serum (NGS) and 0.5% TX-100.
Primary anti-TnI antibody (Chemicon mAb 1691) or anti-FLAG antibody
were diluted in PBS with 0.5% TX-100, and 2% NGS was added to cells
for 1.5 h at room temperature. Cells were then washed with PBS + 0.5% TX-100 and blocked in PBS with 20% normal goat serum in 0.5%
TX-100. Secondary antibody conjugated to Texas Red, (Molecular Probes,
T-862) diluted in PBS with 0.5% TX-100 and 2% NGS was added for
1 h. Coverslips were washed in PBS for 4 × 5 min, mounted,
and sealed. Immunofluorescence labeling of myocytes was viewed on a
Nikon Diaphot 200 microscope fitted with a Noran confocal laser imaging
system and Silicon Graphics Indy work station.
Determination of Single Cardiac Myocyte Force Production--
A
force transducer (Cambridge Technology, Model 403A; sensitivity, 5 µg; 1-99% response time, 1 ms) and moving coil galvanometer (Cambridge Technology 6350 optical scanner) were attached to the chamber via three-way positioners. The temperature of the experimental chamber was controlled (15 °C) using thermoelectric modules (Melcor Inc.) coupled to a recirculating water bath heat sink. The chamber was
positioned on an anti-vibration table. Coverslips were washed several
times in relaxing solution (see below) 2-6 days post-gene transfer,
and single, rod-shaped, cardiac myocytes were then attached to the
glass micropipettes (32). Sarcomere length for each cardiac myocyte was
set at 2.15 µm by viewing the myocyte using light microscopy and then
adjusting the overall length of the preparation by way of three-way
translators. Relaxing and activating solutions contained (in
mM/liter) 7 EGTA, 1 free Mg2+, 4 MgATP, 14.5 creatine phosphate, 20 imidazole, and added KCl to yield a total ionic
strength of 180 mM/liter. Solution pH was adjusted to 7.00 or 6.20 with KOH/HCl. The pCa (i.e. Statistics--
To derive values for the Hill coefficient
(nH) and midpoint from the
tension-pCa relationship (termed K or
pCa50), data were fit using the
Marquardt-Levenberg non-linear least squares fitting algorithm using
the Hill equation in the form: P = (Ca2+)nH/(KnH + (Ca2+)nH), where P
is tension as a fraction of maximum tension
(Po), K is the Ca2+ that
yields one-half maximum tension, and nH is the
Hill coefficient. Analysis of variance was used to examine whether
there were significant differences in pCa50
( Troponin T Isoform Gene Transfer, Expression, and Functional
Assessment--
Isolated cardiac myocytes, transduced with recombinant
virus harboring adult cardiac TnT (aTnT/TnT4) (14) or embryonic cardiac TnT (eTnT, TnT2) (14), were examined for TnT expression, followed by
single myocyte isometric force recordings. On average, eTnT replacement
(calculated as eTnT/(eTnT+aTnT) was about 45% (Fig. 2A). Expression of eTnT was
not detected in myocytes transduced with aTnT (Fig. 2A) or
with reporter vectors (not shown). As demonstrated earlier (25), total
TnT content was not altered in aTnT- or eTnT-transduced myocytes. In
addition, in myocytes membrane-permeabilized just prior to Western
analysis the detection of eTnT was unchanged compared with
membrane-intact myocytes (Fig. 2, A and B). The finding of unchanged total TnT content, together with similar detection
of eTnT in intact and permeabilized myocytes, provides evidence of
sarcomeric replacement of TnT isoforms.
Following TnT gene transfer, steady-state isometric force production
was determined in single cardiac myocytes. Maximum isometric tension
and the slope (Hill coefficient) and position
(pCa50) of the tension-pCa
relationship were not significantly different among the control, aTnT,
or eTnT groups (Fig. 2, C and D).
Troponin I Isoform Gene Transfer, Expression, and Functional
Assessment--
Adult cardiac myocytes were transduced with vectors
harboring rat ssTnI or cTnI expression cassettes. In agreement with
previous work (27-29), ssTnI replacement of endogenous cTnI was
greater than 90% at day six post-gene transfer (Fig.
3, A and B) with no
detected alterations in the expression pattern or stoichiometry of
other sarcomeric proteins (data not shown). In addition, ssTnI or cTnI
expression did not alter total TnI content and is evidence of
stoichiometric replacement of TnI in the sarcomere. To further establish the efficiency and localization of newly expressed TnIs in this setting, we expressed an epitope-tagged TnI in the myocytes (cTnIFLAG). Results showed cTnIFLAG was detected in greater than 95%
of the cardiac myocytes (Fig. 3C) with expression discretely localized to the cardiac thin filament as shown previously (22).
In our previous work (27, 28, 30, 33), we showed that replacement of
>90% of cTnI by ssTnI causes marked increases in the Ca2+
sensitivity of tension development, with an average increase in
Ca2+ sensitivity of 0.26 pCa units. Left unknown
is whether ssTnI replacement can be titrated to <50%, which is
similar to maximum replacement obtained for eTnT (Fig. 2, A
and B), and still cause alterations in Ca2+
-activated tension. Based on the efficiency and synchronization of
myofilament gene transfer (Fig. 3C) and the normal turnover rate of sarcomeric proteins (34), it is possible to construct a
functional dose-response relationship for ssTnI by examining single
myocytes at specific time points after gene transfer. Accordingly, in
myocytes at days 2 and 3 post-gene transfer, ssTnI replacement averaged
at the single myocyte level 14.4 and 20.4%, respectively (Fig. 3,
A and B). Even at these relatively low levels of
replacement there was a significant increase (p < 0.01) in the Ca2+ sensitivity of tension, indicating a
dominant effect of ssTnI to modulate myofilament regulation of
contraction (Fig. 3D). The magnitude increase in
Ca2+ sensitivity of tension was 0.12 and 0.20 pCa units at days 2 and 3, respectively (p < 0.01). For controls, we examined calcium-activated tension in
untreated myocytes and myocytes after cTnI gene transfer at days 2 and
3 in primary culture. Results showed that control and cTnI myocytes
were unchanged and not different from each other (Fig. 3D).
This is in agreement with our earlier finding of stable contractile
function in adult myocytes in short-term primary culture (26). In
separate experiments, we evaluated the extent of ssTnI replacement by
SDS-PAGE/silver staining and through use of a second anti-TnI antibody.
In broad agreement with our main findings (Fig. 3, A and
B), SDS-PAGE showed 27.5% replacement of cTnI by ssTnI at
day 2 and 67% replacement at day 4. Similarly, blots probed with a
second TnI antibody (Fitzgerald) showed 29% replacement of cTnI by
ssTnI at day 2 after gene transfer (Fig. 3, E and
F). Collectively, our findings, using two different TnI
antibodies (Chemicon and Fitzgerald) and SDS-PAGE, demonstrate that the
extent of TnI replacement is between 14-29% at day 2 and that this
extent of TnI replacement achieves nearly the same magnitude shift in Ca2+ sensitivity of tension as we have reported in several
previous studies with > 90% replacement (0.26 pCa
units, Refs. 27, 28, 30, 33).
Tropomyosin Isoform Gene Transfer, Expression, and Functional
Assessment--
Tropomyosin isoforms alone or together with different
TnT isoforms have been postulated to play a role in determining
Ca2+ sensitivity of tension (35-38). In general agreement
with the 5.5-day half-life for Tm (34), gene transfer of Influence of Thin Filament Isoform Expression on pH Sensitivity of
Tension Development--
Acidosis is an important contributor to
cardiac dysfunction during myocardial ischemia (39). Differential thin
filament isoform expression in cardiac development, and among slow and fast skeletal fibers, is thought to play an important role in defining
the magnitude in acidic pH-mediated contractile dysfunction (30, 40).
We therefore compared the relative effects of Tm, TnI, and TnT isoforms
on contractile function at pH 6.20, a level of acidification that
accrues during acute myocardial ischemia (39). In concert with results
obtained under physiological conditions, ssTnI replacement, but not Tm
or TnT isoform replacement, had a significant effect on the
Ca2+ sensitivity of tension under acidic pH conditions
(Fig. 5).
Function of Thin Filament Regulatory Isoforms--
Our results
demonstrate a functional difference among thin filament regulatory
protein isoforms for modulation of Ca2+ sensitivity of
tension. Stoichiometric replacement of < 30% of native
cTnI with ssTnI caused significant alterations in myofilament Ca2+ sensitivity of tension development. By contrast,
30-50% isoform replacement with either eTnT or
In its simplest form, the cardiac thin filament can be considered a
linear array of 24 regulatory units. A regulatory unit is defined as
seven actin monomers, one Tm dimer, and one heterotrimeric troponin
complex consisting of troponin C, troponin T, and TnI (41). There is
considerable evidence that these discrete regulatory units are coupled
functionally. Nearest neighbor and long-range interactions have been
demonstrated among thin filament regulatory units (41-43). Our finding
that 14-29% TnI isoform replacement can cause significant alterations
in function supports the concept of long-range regulatory unit
interactions. We previously showed that TnI replacement is a stochastic
process along the thin filaments, even at early time points after gene
transfer (22). As an example, 14% TnI isoform replacement would
represent an average replacement of 3-4 regulatory units with ssTnI of
a total of 24 in each thin filament. Stated differently, 1 in 7 regulatory units, on average, is switched from cTnI to ssTnI. With
stochastic TnI replacement, a dominant effect of ssTnI is proposed to
extend across 3-4 native cTnI regulatory units from both sides of each
new ssTnI regulated unit. Similar types of long-range interaction have
been inferred by the partial removal of troponin C subunits, where as
little as 5% manipulation in troponin C content has been hypothesized to exert an effect on calcium-activated tension (41, 42). Mechanistically, the near neighbor effects of TnI could be mediated via
tropomyosin. Recent studies on thin filament regulation reveal multiple
transition states/binding interactions of tropomyosin on actin under
the influence of calcium and myosin binding (44). In the present study,
there were further increases in Ca2+ sensitivity as TnI
isoform replacement rose from day 2 to day 3 after ssTnI gene transfer
(Fig. 3). The calcium sensitivity data at physiological and acidic pH
at < 30% TnI replacement are in general agreement with previous
gene transfer studies in which nearly all cTnI had been replaced by
ssTnI (27-29). This suggests apparent saturation of TnI
isoform-dependent functional effects even though 70-80%
regulatory units remained coupled with native cTnI. Collectively, these
results establish a dominant gain-in-function by ssTnI. Because a
change in only about 1 in 7 regulatory units is required to bring about
functional effects, this may impact on strategies to influence
contractile performance in diseased myocardium.
Comparisons to Earlier Work--
Our finding that up to 45%
replacement of Implications--
The finding of a dominant effect of ssTnI
isoform replacement even at low levels on Ca2+ sensitivity
may be useful in designing TnI proteins for addressing cardiac
contractile dysfunction in disease. Based on our results, modified TnI
appears to be the best candidate for improving myofilament Ca2+ sensitivity of tension under acidotic conditions
during acute myocardial ischemia. In failing myocardium, or in
cardiomyopathies associated with altered diastolic Ca2+
levels, TnI chimeras with specified regulatory functions may be
advantageous (27-29). Modified TnIs may also serve as a potential strategy for correcting inherited diseases associated with mutated sarcomeric proteins because these mutations have been associated with
changes in myofilament Ca2+ sensitivity (17, 21). It may be
possible to affect disease progression if these Ca2+
sensitivity shifts are prevented by targeted TnI replacement. Alternatively, pharmaceutical-based strategies to manipulate TnI function may be another approach for future investigation. Finally, although re-expression of embryonic isoforms of TnT and Tm may provide
useful bio-markers of failing adult myocardium, our findings indicate
that these alterations, at least up to 30-50% replacement, do not
appear to contribute to altered calcium-activated tension of the
diseased heart.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and
-Tm isoform ratios are altered during embryonic/fetal myocardial development, with the ratio of
-Tm
gene to
-Tm gene expression increasing from 5:1 in fetal hearts to 60:1 in adult rodent and human myocardium (4). Moreover, differential splicing of cardiac TnT gene transcripts results in
expression of an embryonic TnT (eTnT/TnT2) isoform in the developing myocardium followed by a transition to a more basic, lower molecular weight adult TnT (aTnT/TnT4) in adult myocardium (5-9). In the mammalian heart two distinct TnI genes are expressed (4, 10). Early in
cardiac development the slow skeletal TnI (ssTnI) isoform is expressed.
In the neonatal heart, ssTnI is down-regulated and the cardiac TnI
(cTnI) gene is expressed. In the adult myocardium only the cTnI isoform
is detected. In concert with Tm, TnI, and TnT isoform switching during
development, the cardiac contractile apparatus transitions from being
highly sensitive to being less sensitive to Ca2+ (11, 12).
This process effectively tunes the myofilaments to adaptive alterations
in intracellular calcium handling (11). Additionally, in the diseased
adult myocardium, embryonic TnT and Tm isoforms have been shown to be
re-expressed. For example, in pressure overload hypertrophy
-Tm
expression is increased, and in failing human myocardium the eTnT
isoform is re-expressed (5-7, 9, 13, 14). In myotonic dystrophy
resulting from a CTG expansion in the TnT gene, increases in
CUG-binding protein expression and binding to cardiac TnT
pre-mRNA result in increased production of the longer fetal eTnT
transcript (15, 16). Collectively, these studies provide correlative
evidence that thin filament regulatory isoform composition influences
Ca2+-activated contractile function.
-Tm) or reporter viral gene transfer have further demonstrated the
specificity of this approach in regard to myocyte contractile
structure-function (22, 25, 26, 30). Using this system, adult cardiac
myocytes were systematically transduced with eight different vectors
harboring Tm, TnI, or TnT isoform expression cassettes (Fig. 1). We
then directly compared Ca 2+-activated isometric force
responses of single adult myocytes following specific TnT, TnI, or Tm
isoform replacement. Results demonstrate that ssTnI acts in a dominant
manner with respect to the functional significance of cardiac thin
filament isoforms in modulating regulation: TnI > Tm and
TnT.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Tm (
tropomyosin);
-TmFLAG (C-terminal FLAG epitope-tagged
-Tm);
-Tm (
-tropomyosin), from human cDNAs; eTnT (embryonic cardiac
troponin T/also termed TnT2); and aTnT (adult cardiac troponin T/also
termed TnT4), from rat cDNA (Fig. 1,
A and B). The general strategy and protocols for
shuttle plasmid and vector construction, virus production, and
purification have been described earlier (22, 25, 30, 31). In each
case, transcription was driven by the CMV promoter/enhancer. The
polyadenylation signal derived from SV40.
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Fig. 1.
Vectors and thin filament isoform transitions
in development. A, recombinant Tm, TnI, and TnT
adenoviral vectors. Serotype 5 adenovirus linear structure is shown
with the E1 region deleted and substituted with a eukaryotic expression
cassette in reverse orientation. The eight cDNAs encoding the
regulatory proteins are listed. B, listing of the
developmentally regulated transitions in thin filament regulatory
protein isoforms in the mammalian myocardium.
log
Ca2+) of the relaxing solution was set at 9.0, and the
pCa of the solution for maximal activation was 4.0. At each
pCa, steady-state isometric tension developed, after which
the fiber was rapidly (< 0.5 ms) slackened to obtain the tension
baseline. The myocyte was then relaxed, and the difference between
developed tension and the tension baseline following the slack step was
measured as total tension. To obtain active tension, resting tension
measured at pCa 9.0 was subtracted from total tension. The
myocyte was transferred to relaxing solution after each activation at a
given pCa. Tension-pCa relationships were
constructed by expressing tensions (P) at various submaximal
Ca2+ concentrations as fractions of the tension at maximal
activation, Po (i.e. isometric
tension at pCa 4.0), that bracketed the submaximal activations.
log Ca2+ at half maximal tension),
nH, or maximum tension using
Student-Neuman-Keuls multiple comparison test. A probability level of
p < 0.05 indicated significance.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Troponin T isoform replacement and
Ca2+-activated tension assay. A, Western
blot analysis of TnT isoform expression in intact and
detergent-permeabilized adult cardiac myocytes. Myocytes examined at
day 5-7 post-eTnT and -aTnT gene transfer. Results demonstrate eTnT
expression (TnT 2) after gene transfer of eTnT but not in
control myocytes or in myocytes treated with the aTnT (TnT
4) vector. B, summary of TnT replacement in membrane
intact (I) and permeabilized (P) myocytes. TnT
replacement calculated as eTnT/(eTnT + aTnT).
Values are mean ± S.E., n = 11-17. C,
tension-pCa relationships in single adult cardiac myocytes
at day 5-6 post-TnT gene transfer. Values are mean ± S.E.,
n = 6-9. D, summary of Ca 2+
sensitivity in control, aTnT, and eTnI experimental groups. Maximum
tension, kN/m2 (and Hill Coefficients, nH) for
control, aTnT, and eTnT were 36.4 ± 6.3(2.6 ± 0.4),
30.7 ± 7.8(2.6 ± 0.4), and 32.1 ± 9.6(2.6 ± 0.4). Values are mean ± S.E., n = 6-9.
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Fig. 3.
TnI replacement dose-response relationship in
adult cardiac myocytes. A, Western blot analysis of TnI
isoform expression in adult cardiac myocytes at days 2, 3, and 6 post-ssTnI gene transfer. The soleus muscle sample provides a
positive control for ssTnI expression. B, replacement of
cTnI by ssTnI as a function of time post-gene transfer (ssTnI
replacement = ssTnI/(ssTnI + cTnI)). Values are mean ± S.E.,
n = 6. Asterisks indicate control and
cTnI-transduced myocytes at day 6. C, immunofluorescence
detection of the replacement of native cTnI by epitope-tagged cTnI.
Panels a and c are control, and panels
b and d are following cTnIFLAG gene transfer.
Antibodies are anti-troponin I (panels a and b)
and anti-FLAG (panels c and d). Greater than 95%
of cTnIFLAG-transduced myocytes were positive for cTnIFLAG expression
and localization in the thin filament. D, summary of
pCa50 values at days 2 and 3 post-ssTnI gene
transfer. Maximum tension, kN/m2 (and Hill coefficients,
nH) for control, ssTnI(d2) and ssTnI(d3) were
14.6 ± 6.2(2.2 ± 0.2), 27.4 ± 5.4(3.0 ± 0.4),
15.5 ± 2.8(1.5 ± 0.3). Values are mean ± S.E.,
n = 4-5. Asterisks indicate significantly
different from control, p < 0.01. Gene transfer of
cTnI, with data collected at days 2 and 3 (not different), served as an
additional control. Maximum tension (kN/m2) and Hill
coefficients were 19.6 ± 3.0 and 3.0 ± 0.3. E,
silver-stained SDS-PAGE gel of myocytes post-ssTnI gene transfer.
Lanes 1 and 2, control, day 2; lanes 3 and 4, ssTnI, day 2; lanes 5 and 6,
control, day 4; lanes 7 and 8, ssTnI, day 4. Odd lanes are membrane-intact samples; even lanes
are membrane-permeabilized samples (gave comparable results).
F, TnI antibody comparison. Upper blot is probed
with Chemicon anti-TnI antibody 1691 (same as in panel A);
the same blot was then stripped and reprobed with the Fitzgerald
anti-TnI antibody (lower blot). In both blots, lanes
1-6 are ssTnI, day 2; lanes 7 and 8 are
control, day 2; lanes 9-14 are ssTnI day, 3; lanes
15 and 16 are control, day 3.
-Tm to
adult cardiac myocytes resulted in 30-40% replacement of
-Tm at
day six post-gene transfer (Fig. 4,
A and B). These results were obtained by using
two different anti-Tm antibodies (Fig. 4). In earlier work, total Tm
protein content and the stoichiometry of Tm within the sarcomere were
shown to be unchanged after Tm gene transfer (21, 22). Expression of
-Tm also was not different in intact and permeabilized myocytes
(Fig. 4, A and B), an indication that
-Tm
incorporated into the myofilaments. In functional studies on
permeabilized single cardiac myocytes, neither maximum isometric tension nor the Ca2+ sensitivity of tension (Hill
coefficient/pCa50) were affected by
-Tm,
-TmFLAG, or by
-Tm gene transfer (Fig. 4C legend). Thus, at this level of
-Tm replacement by
-Tm, which is similar to the Tm isoform switching observed during normal myocardial development, myofilament Ca2+ sensitivity is not
significantly altered.
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Fig. 4.
Tropomyosin isoform replacement and cardiac
myocyte function. A, Western blot of Tm isoform
expression in intact and detergent-permeabilized adult cardiac myocytes
after -Tm gene transfer. Myocytes collected at days 5-7 post-gene
transfer. B, summary of
-Tm replacement in control and
- and
-Tm-transduced myocytes.
-Tm replacement calculated as
-Tm/(
-Tm +
-Tm). Values are mean ± S.E.,
n = 7-9. Blots probed with TM311 antibody. CH-1
anti-Tm antibody gave similar results (data not shown) C,
summary of pCa50 values in control and
-Tm-,
-Tm-, and
-TmFLAG-transduced myocytes. The control
pCa50 is different from other control
pCa50s (e.g. Fig. 3) because these
experiments were conducted separately and, over time, the absolute
pCa50s can vary from study to study. Maximum
tension, kN/m2 (and Hill Coefficients, nH) for
control,
-Tm, and
-Tm were 19.8 ± 7.1(1.5 ± 0.1),
15.2 ± 5.8 (1.6 ± 0.1), 10.9 ± 1.6(1.9 ± 0.3).
Values are mean ± S.E., n = 7-9. No significant
differences between groups by analysis of variance.
View larger version (16K):
[in a new window]
Fig. 5.
Summary of altered Ca2+
sensitivity (pCa50) under acidic pH
conditions. Values are mean ± S.E., average of 5 myocytes
per group. Because these data were collected separately over time
(denoted by vertical dashed line) two control groups are
shown, one for the ssTnI and one for the TnT/Tm groups. These controls
values were not different. Measurements obtained at days 2 and 3 for
ssTnI and days 5-7 for TnT/Tm. -Tm, n = 2. Asterisk indicates significantly different from control,
p < 0.01.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Tm had no
significant influence on the Ca2+ sensitivity of tension.
This comparative analysis provides direct evidence that ssTnI has a
dominant modulatory effect on the regulation of contraction in cardiac muscle.
-Tm with the
-Tm isoform has no significant effect
on Ca2+-activated tension is in apparent contrast to
earlier studies on transgenic mice (45). Cardiac bundles isolated from
-Tm transgenic mice show increased Ca2+ sensitivity when
activated under physiological conditions; however, in agreement with
our findings,
-Tm had no significant effect on myofilament pH
sensitivity (46). One factor to consider in comparing these results is
the extent of Tm replacement. In transgenic studies, about 55% or
greater Tm replacement was achieved in vivo (17, 45), a
value greater than we have obtained by Tm gene transfer. Based on
biochemical and transgenic studies (45), it appears that significant
heterodimer formation is necessary for Tm
isoform-dependent effects on myofilament Ca2+
sensitivity. Another issue to consider is that transgenic manipulation of Tm can and does result in organ-level maladaptive growth (19, 20),
and this could impact subsequent assays of myocyte function. Similarly,
eTnT replacement of 40-50% did not alter Ca2+-activated
tension. Previously, correlations have been reported between
developmental/disease-mediated transitions in cardiac TnT isoform
expression and myocardial function (35, 37). As with Tm isoforms,
higher levels of replacement of TnT isoforms may be necessary to detect
changes in myofilament function. In this light, it is interesting to
note that single missense mutations associated with hypertrophic
cardiomyopathy (HCM) and placed in eTnT can significantly affect
calcium sensitivity, even at very low levels (about 8%) of TnT
replacement (25). Similarly, HCM mutant Tms produce alterations in
function at replacement levels comparable with the
-Tm replacement
achieved in the present study (21). There are 39 amino acid differences
between
- and
-Tm, including many in the C terminus thought to be
critical for Tm and TnT interactions (47). In our functional assay,
these 39 amino acid differences appear less significant than
HCM-associated single missense mutations in Tm. However, regarding the
developmental regulation of thin filament isoform expression, it is
apparent that TnI has a much more dominant effect than TnT or Tm
isoforms on developmental tuning/modulation of myofilament
Ca2+ sensitivity. We cannot exclude the possibility that if
higher levels of TnT or Tm replacement could be achieved in this system this would then cause alterations in the Ca2+ sensitivity
of tension. This result, if obtained, would suggest that the threshold
for thin filament isoforms to have a modulatory effect on regulation is
markedly higher for Tm and TnT than for TnI isoforms.
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FOOTNOTES |
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* This work is supported by grants from the National Institutes of Health and the American Heart Association (to J. M. M.).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. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Physiology, 7730 Medical Science Bldg. II, University of Michigan, Ann Arbor, MI 48109-0622. Tel.: 734-763-0560; Fax: 734-647-6461; E-mail: metzgerj@umich.edu.
Recipient of a Scientist Development grant from the American
Heart Association.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212601200
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ABBREVIATIONS |
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The abbreviations used are: TM, tropomyosin; TnT, troponin T; TnI, troponin I; ssTnI, slow skeletal TnI; cTnI, cardiac TnI; cTnIFLAG, C-terminal, FLAG epitope-tagged cTnI; eTnT, embryonic eTnT; aTnT, adult TnT; NGS, normal goat serum; TX-100, Triton X-100.
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