Download Coexpression of Cardiac titins

K. Trombitás, Y. Wu, D. Labeit, S. Labeit and H. Granzier
Am J Physiol Heart Circ Physiol 281:1793-1799, 2001.
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Am J Physiol Heart Circ Physiol
281: H1793–H1799, 2001.
Cardiac titin isoforms are coexpressed in the
half-sarcomere and extend independently
K. TROMBITÁS,1 Y. WU,1 D. LABEIT,2 S. LABEIT,2 AND H. GRANZIER1
1
Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State
University, Pullman, Washington 99164-6520; and 2Anesthesiology and Intensive Operative
Medicine, University Hospital Mannheim, Mannheim, Germany 68135
Received 22 May 2001; accepted in final form 6 July 2001
diastole; elasticity; structure; mechanics; physiology
consists of thin filaments attached to Z lines, thick filaments located in
the center of the sarcomere, and titin filaments that
span from the Z line to the middle of the sarcomere (8).
The thin and thick filaments develop active force,
whereas titin filaments develop passive force (6). The
force of titin is derived from the I-band region of the
molecule that extends while the sarcomeres are
stretched (17). The I-band region of cardiac titin has a
complex sequence with three distinct elements that
extend along the physiological sarcomere length (SL)
range of the heart: 1) the PEVK segment, rich in
proline (P), glutamic acid (E), valine (V), and lysine (K)
residues; 2) serially linked Ig-like domains that make
up tandem Ig segments; and 3) the N2B element (4,
10–12).
Most of the thin and thick filament-based muscle
proteins exist as multiple isoforms, either derived from
different genes or from differential splicing of the same
gene that are expressed in a developmental- and dis-
THE STRIATED MUSCLE SARCOMERE
Address for reprint requests and other correspondence: H. Granzier, VCAPP, Washington State Univ., Pullman, WA 99164-6520
(E-mail: [email protected]).
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ease-specific manner (13). Recent work (4) indicates
that titin is derived from a single gene on chromosome
2 (region 2q31) and that extensive exon shuffling in the
I-band region of the molecule results in a wide range of
isoforms. In the heart, titin transcripts are processed
by two distinct splice routes, which vary within their
extensible I-band regions: 1) the so-called N2B titins
containing the N2B element and 2) N2BA titins containing both the N2B and N2A elements (2, 4) (see Fig.
1). N2BA titins contain a much longer PEVK segment
than do N2B titins (600–800 vs. 163 residues), as well
as an additional tandem Ig segment (4).
Recent sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) studies (2) of myocardium
isolated from various species indicate that the expression level of N2B and N2BA cardiac titins varies from
predominantly N2B (rat and mouse) to predominantly
N2BA (bovine atrium), with many species (including
human) coexpressing isoforms at intermediate levels.
Changes in titin isoform expression may be important
during heart disease because alterations in the titin
isoform expression level have been observed in pacing
tachycardia heart failure in the dog (1). It is not known
whether sarcomeres are isoform pure and whether
coexpression is the result of mixtures of sarcomeres
that express different isoforms or whether coexpression occurs at the level of the half-sarcomere. If the
latter were to be the case, the question arises of
whether different isoforms contained within the halfsarcomere interact. Solving these issues is important
for understanding the functional significance of coexpressing isoforms and its alterations in heart disease.
Immunofluorescence and differential staining of the
sarcomere with isoform-specific antibodies cannot be
used to study coexpression because the full N2B sequence is contained within N2BA titin (4). Here we
used immunoelectron microscopy (IEM) with the N2B
antibody, raised against a sequence found in both isoform types, that labels closer to the Z line in N2BA titin
than in N2B titin (see RESULTS). Thus the N2BC antibody and IEM are ideal for the study of cardiac titin
isoform expression at the sarcomeric level. Our results
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Trombitás, K., Y. Wu, D. Labeit, S. Labeit, and H.
Granzier. Cardiac titin isoforms are coexpressed in the
half-sarcomere and extend independently. Am J Physiol
Heart Circ Physiol 281: H1793–H1799, 2001.—Titin, the
third myofilament type of cardiac muscle, contains a molecular spring segment that gives rise to passive forces in
stretched myocardium and to restoring forces in shortened
myocardium. We studied cardiac titin isoforms (N2B and
N2BA) that contain length variants of the molecular spring
segment. We investigated how coexpression of isoforms takes
place at the level of the half-sarcomere, and whether coexpression affects the extensibility of the isoforms. Immunoelectron microscopy was used to study local coexpression of
isoforms in a range of species. It was found that the cardiac
sarcomere of large mammals coexpresses N2B and N2BA
titin isoforms at the level of the half-sarcomere, and that
when coexpressed, the isoforms act independently of one
another. Coexpressing isoforms at varying ratios results in
modulation of the passive mechanical behavior of the sarcomere without impacting other functions of titin and allows
for adjustment of the diastolic properties of the myocardium.
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COEXPRESSION OF CARDIAC TITIN ISOFORMS
bovine) coexpressed N2B and N2BA titins, whereas the
bovine atrium is nearly N2BA pure. These findings are
consistent with earlier results (2). To study the coexpression of titin, we used IEM with an antibody against
the COOH-terminal end of the N2B element (N2BC).
Fig. 1. Domain structure of I-band regions of cardiac N2B and N2BA
titins and location of binding sites of used antibodies (4) [T12, N2BN,
N2BC, N2A, I84–86, and I109–111]. Two N2BA splice pathways are
shown, representing the pathway with the lowest (12) and highest
(25) number of Ig domains identified so far in the titin gene sequence
(4). Red, Ig domains; white, fibronectin domains; yellow, PEVK; blue,
unique sequence.
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show that coexpression of N2B and N2BA isoforms
takes place at the level of the half-sarcomere and that
different isoforms expressed in the half-sarcomere extend independently. We propose that coexpression of
isoforms at the level of the half-sarcomere allows for
tuning of the passive properties of cardiac muscle.
METHODS
Muscles. Hearts were excised from ;9-wk-old BALB/C
male mice, mongrel dogs (;20 kg), and cows (;18 mo old)
obtained from slaughterhouses. The free wall of the left
atrium and left ventricle were cut into pieces and placed into
oxygenated Krebs solution (19). Muscle strips were dissected
in the direction of the fibers, while stretch of the preparations
was minimized. Muscles were skinned in relaxing solution
(6) containing 1% wt/vol Triton X-100.
SDS-PAGE. Muscles were quick-frozen in liquid nitrogen,
pulverized to a fine powder, rapidly solubilized, and then
analyzed with the use of SDS-PAGE (6). The gels were
scanned as described in an earlier study (6).
Titin antibodies. Antibodies that were used and the locations of their epitopes in titin are shown in Fig. 1. Titin cDNA
fragments coding for the I-band epitopes I24/I25 to I109-I111
were isolated by polymerase chain reaction from total human cDNAs. All primer sequences were derived from European Molecular Biology Laboratory data library accessions
X90568/X90569 and are described in detail elsewhere (4).
The amplified cDNA fragments were subcloned into modified
pET9D vectors and fusion peptides with NH2 terminal His6tags were purified from the soluble fractions by nickel chelate
affinity chromatography. Antibodies to the respective peptides were raised in rabbits and were affinity purified (4). The
titin antibody T12 recognizes the I2/I3 repeats (14) and was
purchased from Boehringer (Indianapolis, IN).
IEM. Skinned muscles while in relaxing solution were
stretched to different lengths, fixed, immunolabeled, embedded, and processed for IEM essentially as described (5). The
mid-Z-line to mid-epitope distances were measured from IEM
negatives after high-resolution scanning (model UC-1260,
UMAX) and digital image processing using custom-written
macros for the NIH Image 1.6. For spatial calibration, the
magnification of the microscope was used.
RESULTS
Gel electrophoresis of left ventricular myocardium
(Fig. 2A) revealed that the mouse expressed predominately N2B titin and that large mammals (dog and
AJP-Heart Circ Physiol • VOL
Fig. 2. A: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) of myocardial tissues from left ventricle (V) of mouse,
dog, and bovine and left atrium (A) of bovine. Mouse ventricle
contains predominantly N2B titin. Dog and bovine ventricle (BV)
express N2B and N2BA2 titins, whereas bovine atrium expresses
predominately N2BA titin. T2 is a degradation product of titin. MHC:
myosin heavy chain. B: examples of sarcomeres labeled with the
N2BC antibody. Note that dog and BV sarcomeres contain two N2BC
epitopes: one near the Z line (A) and the other closer to the A band
(B), whereas the other specimen contains a single epitope (see Fig. 3).
Asterisk denotes labeling with I109–111 (stains at A-band edge). See
text for details. Scale bar, 1.0 mm.
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COEXPRESSION OF CARDIAC TITIN ISOFORMS
Fig. 3. Distance between N2BC epitopes and middle of Z line in BV
sarcomeres. Also shown are the N2BC results of rat ventricle (RV)
and bovine atrium (BA) (see Refs. 16 and 18). Results of these earlier
studies were pooled in 0.1-mm sarcomere length (SL) bins, and
means 6 SD of SL and epitope distances are plotted. Lines are
second-order polynomial fits of the BV data. F, N2BC antibody in RV;
■, N2BC antibody in BA.
AJP-Heart Circ Physiol • VOL
N2B and N2BA titins (Fig. 1). Representative micrographs are shown in Fig. 4, A–D, and a graph of
epitope to Z-line distance is shown in Fig. 4E. The
various epitopes in the bovine ventricle were assumed
to result from coexpressed N2B and N2BA isoforms.
The epitopes that demarcate the PEVK segment and
N2B unique sequence of N2B titin are shown in Fig. 4F
and those of N2BA titin in Fig. 4G. With the use of
these epitopes, we determined the lengths of the PEVK
segments and N2B unique sequences. Our results
show that the two PEVK segments that can be discerned in the bovine ventricle sarcomere respond differentially to sarcomere stretch (red and green symbols
in Fig. 5A), as do the two N2B sequence elements (red
and green symbols in Fig. 5B).
We overlaid the bovine ventricular results with those
obtained earlier in the rat ventricle and bovine atrium
(16, 18). The bovine ventricular PEVK segment that
extends most steeply with SL (Fig. 5A, red symbols)
behaves like the PEVK of the bovine atrium (Fig. 5A),
whereas the other PEVK segment (Fig. 5A, green symbols) extends like the PEVK of the rat ventricle (Fig.
5A). The bovine ventricular N2B unique sequence that
extends most steeply with SL (Fig. 5B, red symbols)
behaves like the unique sequence of the rat ventricle
(Fig. 5B) and the one that extends less steeply behaves
like the unique sequence of the bovine atrium (Fig. 5B).
As further explained below, these findings indicate
that the bovine ventricle coexpresses, at the level of the
half-sarcomere, N2B and N2BA isoforms that extend
independently of one another.
Although staining with the N2BC antibody typically
revealed two epitopes of similar intensity in the I band
of bovine ventricular sarcomeres, on occasion, one of
the epitopes dominated. Figure 6A, bottom, shows an
example of such atypical labeling pattern (seen in ,1%
of the examined sarcomeres). Variation in staining was
also found in other species. For example, in the rat
ventricle, the overwhelming majority of sarcomeres
(.99%) contained a single epitope near the A band, but
on occasion there was a second epitope more toward
the Z line (Fig. 6B, bottom). Because both epitopes are
derived from a single antibody, variation in diffusion of
the antibody into the tissue is unlikely to underlie the
variable staining patterns. Instead, they may derive
from variation in the expression patterns of N2B and
N2BA isoforms.
We also studied variation in isoform expression in
the bovine atrium and were unable to find clearly
separated N2BC epitopes, suggesting that bovine
atrium expresses predominantly N2BA titins. When
labeling bovine atrium with the N2A antibody we
noted that long sarcomeres often contained closely
spaced double epitopes (Fig. 6C). The separation of the
epitopes did not vary markedly along the studied SL
range (;2.0–2.5 mm), with a mean separation of 68 6
13 nm (n 5 24). These findings can be explained by
assuming that bovine atrium coexpresses N2BA isoforms and that the two N2A epitopes result from differences in the middle tandem Ig segment contained
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This element is found in both isoforms but is closer to
the A band in N2B than N2BA titin (see Fig. 1 and
DISCUSSION). Immunoelectron micrographs of cardiac
sarcomeres from a range of species labeled with N2BC
are shown in Fig. 2B. At a given SL, sarcomeres from
myocardium that expresses predominately N2B titin
contain a single epitope toward the A band (Fig. 2B,
top) and sarcomeres from myocardium that expresses
predominately N2BA titin contain a single epitope
closer to the Z line (Fig. 2B, bottom). Thus the N2BC
antibody can be used to study coexpression of isoforms
in the I band because coexpression will result in two
epitopes, one near the A band derived from N2B titin
and one closer to the Z line derived from N2BA titin.
When myocardium of species that coexpress isoforms
(as deduced from SDS-PAGE) was labeled with N2BC,
two I-band epitopes were typically found (see Fig. 2B,
top middle and bottom middle; the near Z line and near
A-band epitopes are referred to as A and B epitopes,
respectively). The finding of two epitopes is consistent
with the notion that the sarcomere of ventricular myocardium of large mammals coexpresses isoforms of
titin.
We studied the location of the two N2BC epitopes in
the bovine ventricular sarcomere and determined the
dependence of the epitope-Z-line distance on SL. The
two epitopes are merged at a SL of ;1.8 mm but they
separate as SL increases and are ;0.2 mm apart at a
SL of 3.0 mm (Fig. 3). Figure 3 also shows results (16,
18) with the N2BC antibody in rat ventricle and bovine
atrium. The N2BC(B) epitope of bovine ventricle superimposes with rat ventricular results and the N2BC(A)
epitope with the results of the bovine atrium. These
findings suggest that the bovine ventricle coexpresses
two isoforms, one that behaves like the N2B isoform of
the rat and the other like the N2BA isoform of the
bovine atrium.
We also determined in the bovine ventricle the
lengths of the PEVK and N2B unique sequences, by
using antibodies that demarcate the PEVK and N2B
unique sequence domains in the extensible region of
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Fig. 4. A–D: example of BV sarcomeres labeled with indicated anti-titin
antibodies. (Scale bar, 1.0 mm.) E: scattergram of epitope to Z-line distance vs.
SL. Labeling patterns are assumed to
be derived from N2B and N2BA isoforms that are coexpressed; their
PEVK and N2B unique sequence segments are shown in 4F (N2B titin) and
G (N2BA titin). Lines in F and G are
the second-order polynomial fits of the
data in E.
within these isoforms (Fig. 1). To test whether different
N2BA bands can be detected on gels, we performed
high-resolution electrophoresis. This revealed a doublet in the N2BA region (Fig. 6D), the bottom band of
which comigrated with the single N2BA band of the
bovine ventricle (Fig. 6E). We conclude that the bovine
atrium coexpresses N2BA isoforms that vary considerably in molecular mass.
DISCUSSION
Work at the mRNA and protein levels has revealed
that cardiac muscle contains two classes of titin isoAJP-Heart Circ Physiol • VOL
forms, the N2B and N2BA titins (2, 4). Our goal was to
investigate whether coexpression occurs at the level of
the smallest functional unit of muscle, the half-sarcomere, and how coexpression affects the extensibility
of the isoforms. We used the N2BC antibody, raised
against a sequence found in both isoform types, that
labels closer to the Z line in N2BA titin than in N2B
titin. This different sarcomeric location results from
the large number of additional Ig domains and PEVK
residues that are present in N2BA titin, COOH-terminal of the N2BC epitope (4). These extra sequences
greatly increase the length of the extensible region of
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COEXPRESSION OF CARDIAC TITIN ISOFORMS
N2BA titin, and a given SL can therefore be reached
with a fractional extension of the extensible region that
is lower in N2BA than in N2B titin. Hence, the N2BC
epitope will be closer to the Z line in N2BA than in N2B
titin, and the N2BC antibody can be used to study
coexpression of titin isoforms within the sarcomere
(Fig. 7A).
When myocardium that coexpresses N2B and N2BA
isoforms was labeled with N2BC, two epitopes per
half-sarcomere were seen, one near the Z line in a
location that is similar to that of N2BA expressing
bovine atrium and another closer to the A band in a
location that is similar to that of N2B-expressing rat
sarcomeres (Fig. 3). For the first time, these findings
provide evidence that the cardiac sarcomere of large
mammals coexpresses isoforms of titin at the level of
the half-sarcomere. Thus, not only is the I-band region
of cardiac titin a complex molecular spring with three
distinct subsegments (5, 8), an additional level of complexity results from isoforms that contain length variants of these spring segments that are coexpressed side
by side.
Variation in coexpression patterns. We found that
the expression level of titin isoforms in sarcomeres
obtained by IEM was typically similar to that in muscle
obtained by using SDS-PAGE and Western blotting (2).
However, deviations were found as well. For example,
the overwhelming majority of sarcomeres in rat myocardium contain a single N2BC epitope (as expected
from gel results), but on occasion, sarcomeres were
found with two epitopes (Fig. 6B), suggesting that both
isoform types were coexpressed. Furthermore, in the
Fig. 6. Unusual N2BC staining patterns. A, top:
typical labeling pattern with two I-band epitopes (A
and B) of BV sarcomeres; bottom, atypical sarcomere with strong N2BC(A) epitope and barely visible
N2BC(B) epitope (arrow). B, top: typical labeling pattern with one epitope in I band for N2BC-labeled rat
sarcomeres; bottom, unusual pattern with two
epitopes. Asterisk denotes labeling with I109–111.
C: example of bovine atrial sarcomeres labeled with
the N2A antibody. Note two closely spaced epitopes
at bottom right corner. D: SDS-PAGE of BA, BV,
and RV. Note in BA two closely spaced bands in the
N2BA region of gel. E: densitometry of gel revealing
two N2BA peaks in BA (solid lines), BV (dashed
lines), and RV (gray solid lines). The lower mobility
N2BA peak of BA comigrates with the single N2BA
of BV. Scale bars in A–C, 1.0 mm.
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Fig. 5. SL dependence of end-to-end lengths of PEVK (A) and N2B
unique sequence (B) of the BV, superimposed with results of RV and
BA. Earlier RV and BA data (16, 18) calculated in Fig. 3. Lines are
second-order polynomial fits of the BV data.
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COEXPRESSION OF CARDIAC TITIN ISOFORMS
bovine ventricle, two N2BC epitopes of similar intensity were typical (consistent with gel results), but on
occasion, the near-Z-line epitope derived from N2BA
titin dominated (Fig. 6A). Thus SDS-PAGE and Western blotting provide insights in the average expression
level of the isoforms within muscle, whereas the IEM
method shows that expression patterns can locally
deviate from this average. Deviations in expression
patterns are likely to reflect local differences in demands on the functions of titin that are different from
the tissue average.
Coexpression of N2BA isoforms. At the mRNA level,
seven N2BA isoforms have been characterized that
differ in the length of the middle tandem Ig segment (4)
(the extremes are shown in Fig. 1). The double N2A
epitopes that we found (Fig. 6C) suggest coexpression
of N2BA isoforms in the bovine atrium. Considering
that the average Ig domain spacing of tandem Ig segments is ;5 nm (16, 18), the N2A epitope separation of
68 nm (see RESULTS) suggests a ;14-Ig domain difference between the large and the small N2BA isoforms
expressed in bovine atrium. A 14-Ig difference is expected to result in a molecular mass difference of ;140
kDa (;10 kDa per Ig domain), a difference that in the
MDa molecular mass range may be just detectable on
gels (7). Consistent with this are SDS-PAGE results
that showed closely spaced N2BA bands in the bovine
atrium (Fig. 6, D and E). Thus both SDS-PAGE and
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Fig. 7. A: schematic representation of sarcomere coexpressing
N2B and N2BA titins with PEVK and N2B unique sequences
that extend independently. B: titin-based passive tension-SL relations of isoform pure myocardium [see (19)]. N2B expressing
sarcomeres develop much higher passive tensions than N2BAexpressing muscle. By coexpressing isoforms at varying ratios, an
intermediate passive tension level can be obtained. See text for
details.
IEM results support that members of the N2BA isoform class are coexpressed in the bovine atrium.
Is there interaction between coexpressed isoforms?
Understanding the functional significance of coexpression of isoforms at the level of the sarcomere
requires insights into whether or not the isoforms act
independently of one another. Sequences contained
within the extensible region of the isoforms may
interact with those of other isoforms in their vicinity
or, alternatively, titin-binding proteins may laterally
link the different isoforms and influence the elastic
properties of titin. Our results indicate that the
extensibility of PEVK and N2B unique sequences in
isoform-pure sarcomeres is indistinguishable from
that in isoform-mixed sarcomeres (Fig. 5, A and B).
Thus lateral interactions between the isoforms are
either weak or absent and the isoforms extend independently of one another. Independent behavior is
shown schematically in Fig. 7A.
Functional significance. Passive force levels intermediate between that of myocardium that expresses predominately N2BA or N2B titin (Fig. 7B) can, in theory,
be achieved by varying the number of titin molecules
per sarcomere. However, this would also influence
functions performed by the inextensible regions of
titin. These regions play roles in construction and
maintenance of Z lines and M lines, thick filament
length control, and binding of ligands [for a review, see
Gregorio et al. (8)]. Thus varying the number of titin
molecules to achieve a certain passive force level may
be undesirable because other roles of titin would be
impacted as well. This issue does not exist when passive force levels are tuned via coexpressing isoforms
because the inextensible regions of the isoforms are the
same. Any force level intermediate between that of
isoform-pure sarcomeres may be obtained by varying
the expression ratio of isoforms (see also Fig. 7B) without impacting other functions of titin.
It is likely that the extensible region of titin performs functions that go beyond passive force development and any of these may be modulated via
coexpressing isoforms. Recent evidence (3) suggests
that titin-based passive force influences active force
development, and the effect of titin on active force
may be regulated by coexpressing isoforms at varying ratios. Furthermore, the extensible region of titin
is likely to contain isoform-specific binding sites for
ligands. For example, a binding site for the protease
P94 is found in the extensible region of N2BA titin
only (15), and by varying the expression level of
N2BA titin, the role of P94 in protein turnover may
be modulated. Thus the existence of trace amounts of
N2BA titin on a N2B titin background that is present
in, for example, the rat (Fig. 5B), may not to be
significant for passive tension development, but
could be important for regulating proteolysis.
In conclusion, the half-sarcomere of cardiac muscle
can express a complex pattern of titin isoforms. Coexpressing isoforms either belonging to the same isoform
class (bovine atrium) or belonging to different classes
(ventricular myocardium of most large mammals) is a
COEXPRESSION OF CARDIAC TITIN ISOFORMS
common occurrence in the cardiac sarcomere of large
mammals. We propose that coexpressing isoforms at
the level of the half-sarcomere allows for tuning of the
passive properties of the sarcomere without impacting
other functions of titin. Titin-based passive force underlies a majority of the passive force of the myocardium, except for toward the upper limit of the physiological SL range where collagen dominates (6, 19), and
varying the coexpression ratio of titin may be an effective mechanism for modulating the passive properties
of the myocardium.
This study was supported by Deutsche Forschungsgemeinschaft
Grants La668/4-2 and 6-1 (to S. Labeit) and by National Heart, Lung,
and Blood Institute Grants HL-61497 and HL-62881 (to H. Granzier).
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