© 1995 American Heart Association, Inc.
Articles |
The Mechanically Active Domain of Titin in Cardiac Muscle
From the Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology (H.G.), Washington State University, Pullman; the Department of Medical Biochemistry (J.-P.J.), University of Calgary (Canada); and the Central EM Laboratory (K.T.), University Medical School of Pécs (Hungary).
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Abstract One of the main contributors to passive tension of the myocardium is titin. However, it is not exactly known what portions of this
1 µm-long molecule are anchored in the
sarcomere (hence, are rendered inelastic) and what portions are elastic
(hence, are mechanically active in developing passive tension). We
assessed the length of the elastic domain of cardiac titin by
ultrastructural and mechanical methods. Single cardiac myocytes were
stretched by various amounts, and while in the stretched state, they
were processed for immunoelectron microscopy. Several monoclonal
anti-titin antibodies were used, and the locations of the titin
epitopes in the sarcomere were studied as a function of sarcomere
length. Only a small fraction (5% to 10%) of the 1000-nm-long
molecule behaved elastically under physiological
conditions. This mechanically active domain is located close to the A/I
junction, and its contour length when unstretched is estimated at 50
to 100 nm. In sarcomeres that are slack (length 1.85 µm), the
mechanically active domain is folded on top of itself, and the length
of the domain reaches an elastic limit of 550 nm in sarcomeres that
are 2.9 µm long.
Key Words: cardiac myocytes • immunoelectron microscopy • titin • passive tension • elasticity
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When passive muscle is stretched, it develops tension (known as passive tension) that opposes the stretch and restores the original length of the muscle after release. The molecular basis of passive tension may lie in the organization and properties of the protein titin (also known as connectin) found in striated muscles (for reviews, see References 1 through 31 2 3 ). In skeletal muscle, a single titin molecule extends from the Z line to the M line of the sarcomere, a distance of
1 to 2 µm. While the segment of
the titin molecule found in the I band behaves elastically when
sarcomeres are stretched, the A-band segment is
inelastic.4 5 6 The I-band domain of titin has been proposed
to underlie passive tension of muscle by functioning as a molecular
spring that develops a restoring force when stretched.1
Consistent with this proposal, passive tension is greatly
reduced after titin has been destroyed.7 8 9 10 Most of our knowledge about titin and passive tension has been obtained from studies using skeletal muscle. However, titin and passive tension are likely to be more important in the myocardium. Passive tension is part of the heart's wall tension that, during diastole, determines to what extent the heart will be filled. In support of this notion, the level of expression of titin in hearts of human patients with dilated cardiomyopathy has been reported to be reduced.11 Titin may thus play a critical role in the normal pumping function of the heart. In a recent study, we showed that over the working range of sarcomeres in the heart, titin is one of the main contributors to passive tension.7 Further, passive tension developed by titin increases much more steeply with sarcomere length in cardiac muscle than it does in skeletal muscle. It has been suggested that the steeper tension increase of cardiac muscle is a result of the smaller titin size variant expressed by cardiac muscle.7 12 The small size variant will result in a shorter I-band domain of titin, giving rise to a larger strain of this domain and thus a higher passive tension for a given degree of sarcomere stretch.7 A basic assumption in this proposal is that the full I-band domain of titin is elastic in both skeletal and cardiac muscle. However, recent studies on skeletal muscle indicate that only about half of the I-band domain is elastic in this muscle type.4 6 13 Thus, in order to compare the passive tension of cardiac and skeletal muscles and to understand better the passive tension of the heart in general, it is important to know the length of the titin domain that is elastic and mechanically active in developing passive tension.
We investigated the elastic behavior of different segments of the titin molecule by using immunoelectron microscopy on rat cardiac myocytes that had been stretched and labeled with anti-titin antibodies. Only a relatively small fraction (25% to 50%) of the unstrained I-band domain of titin turned out to be elastic and mechanically active in developing passive tension. We estimate that the unstretched mechanically active domain has a contour length of only 50 to 100 nm, or 5% to 10% of the total length of the titin molecule. In sarcomeres that are slack, we found evidence that the mechanically active domain is highly folded on top of itself.
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Materials and Methods |
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Preparations
Cardiac Myocytes
Single myocytes were isolated from the ventricles of rat (male Sprague-Dawley,
250 g) hearts using the method of Granzier
and Irving.7 To prevent titin degradation, cells were
extensively washed before skinning, and all solutions contained
protease inhibitors.7
Skeletal Muscle Fibers
Single muscle fibers were dissected from the semitendinosus
muscle of adult male New Zealand White rabbits (weight, 3 kg).
Single fibers were mechanically skinned and glued to small aluminum
clips. For further information, see References 7 and 147 14 .
Mechanics
Cardiac Myocytes
The mechanical setup has been described in detail in Granzier
and Irving.7 Briefly, cells were added to a
temperature-controlled flow-through chamber (volume, 150
µL) that was attached to the microscope stage, and a single cell was
glued at one end to the motor and at the other end to the force
transducer. Sarcomere length was measured by using digital image
analysis and Fourier transformation of videotaped cell images.
For immunolabeling experiments, the cells were glued in the stretched
state to the removable coverslip that functioned as the bottom of the
chamber, by first applying two small droplets of glue to the coverslip,
100 µm apart, and then lowering the ends of the cell into this
glue. The cells were surrounded by a shallow ring glued to the
coverslip, which served as a minichamber (volume, 10 µL) for
immunolabeling, fixing, and embedding of the cells.
Skeletal Muscle Fibers
The setup and the sarcomere length measuring technique have been
described in detail by Granzier and Irving.7
Protocols
Mechanical protocols were similar to those in Granzier and
Irving.7 Because rat cardiac myocytes and rabbit
semitendinosus muscle reach an elastic limit at a sarcomere length of
2.9 µm and 4.5 µm, respectively, which if exceeded may result
in permanent mechanical damage,7 14 the stretch amplitude
was kept below those limits. The obtained passive
tension–sarcomere length curves were highly reproducible. All
mechanical experiments were conducted at 20°C.
Immunolabeling and Electron Microscopy
Cardiac myocytes were fixed for 20 minutes in freshly prepared
3% paraformaldehyde in PBS containing (mmol/L) KCl
2.7, KH2PO4 1.5, NaCl 137,
Na2HPO4 8.0, and EGTA 2, pH 7.2. After washing
the cells three times with PBS for 30 minutes each, cells were blocked
for 30 minutes in PBS/0.5% BSA. Cells were then incubated 24 to 48
hours with anti-titin monoclonal antibodies in PBS/BSA. The
following antibodies were used: T12 (2 µg/mL), 9D10 (20 µg/mL), and
Ti-102 (ascites 20x diluted). T12 is an antibody that was developed by
Dr Weber (Max Planck Institute for Biophysical Chemistry, Goettingen,
Germany) and is against skeletal muscle titin.4 T12 has
been shown to be titin specific and to cross-react with cardiac
titin.4 It is commercially available from
Boehringer (No. 1248-634). The antibody 9D10 is against bovine
cardiac titin and was developed by Dr Greaser (Muscle Biology
Laboratory, University of Wisconsin). It has been shown to be titin
specific.15 We obtained 9D10 from the Developmental
Studies Hybridoma Bank maintained by the Department of Biological
Sciences, University of Iowa, Iowa City. Finally, Ti-102 is a
titin-specific monoclonal antibody against cloned rat cardiac titin
class II motifs16 and was developed by Dr Jin (Department
of Medical Biochemistry, University of Calgary). After washing cells
three times with PBS/BSA, they were incubated for 24 to 48 hours in
secondary antibody in PBS/BSA: for T12, rabbit anti-mouse IgG; for
9D10, goat anti-mouse IgG, IgA, and IgM; and for Ti-102, rabbit
anti-mouse IgG whole molecule. In several experiments, the
secondary antibody was nanogold Fab anti-mouse IgG (No. 2002,
Nanoprobes Inc). Unbound antibody was removed by washing with PBS.
Solutions contained 100 µg/mL leupeptin and were kept at 4°C.
Cells were fixed with glutaraldehyde/tannic acid,
osmicated, and embedded in araldite (compare with Reference 66 ). The
osmification step was omitted when nanogold Fab anti-mouse IgG was
used. Instead, a silver-enhancement step was introduced (K.
Trombitás, unpublished data, 1995) by using HQ silver (Nanoprobes
Inc) as per the manufacturer's protocol. The solutions for
immunolabeling, fixing, and embedding were added while the cell
remained glued to the glass coverslip inside a 10-µL chamber. Cells
were embedded by inverting an araldite-filled beem capsule over the
ring glued to the coverslip. After polymerization, the coverslip was
removed by exposure to liquid nitrogen followed by an exposure to
60°C H2O. Ultrathin sections were cut with a Sorvall
MT-2M ultramicrotome. They were stained with potassium permanganate and
lead citrate and studied with a Hitachi H-600 electron microscope.
Distances from the Z line to the epitope were obtained from electron
micrographs. For spatial calibration, the A-band width (1.6 µm) was
used as a standard.
Gel Electrophoresis
Cells and skeletal muscle were solubilized and electrophoresed
by using 2% to 12% acrylamide gradient gels that were
stained with either Coomassie blue or ammoniacal silver
stain.7
Statistics
Results of the present study are given as the mean±SD,
unless indicated otherwise. Significance in selected
parameters was examined by Student's t test
(P<.05).
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Results |
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Titin in Cardiac Myocytes
To assess the integrity of titin in isolated cardiac myocytes, we measured the passive tension–sarcomere length relation of cardiac cells and compared it with results from skeletal muscle fibers. Because passive tension is derived not only from titin but also from intermediate filaments (IFs), we dissected the contribution of titin to passive tension using a KCl/KI treatment (compare with Reference 77 ), which abolishes titin-based tension while leaving that of IFs intact.7 12 17 18 We found that both IFs and titin developed much higher levels of tension in cardiac myocytes than in skeletal muscle fibers (Fig 1
). At a sarcomere length of
2.3 µm, tension developed by IFs and titin was 10 to 20 times higher
in cardiac cells than in skeletal muscle fibers. Titin-based
passive tension increased exponentially in both muscle types (Fig 1).
The increase was steepest in cardiac cells. The high levels of passive
tension developed by cardiac titin confirm earlier
findings7 and indicate that titin is not degraded by our
cardiac myocyte isolation procedure.
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We also electrophoresed solubilized cardiac myocyte proteins side by
side with rabbit skeletal muscle. Cardiac titin consisted of a doublet:
T1 and T2 (inset, Fig 1
). T1 of rat
cardiac muscle migrated much further on the gels than did
T1 of skeletal muscle, confirming the earlier finding that
rat cardiac muscle expresses a low-molecular-mass size variant
of titin.7 T2 is generally considered a
product of degradation of the parent molecule,
T1, a process occurring during sample
preparation.1 The level of degradation in rat cardiac
myocytes (inset, Fig 1) is typical of the degradation seen in most
skeletal muscles,12 indicating that
collagenase digestion of the heart did not result in any
additional titin degradation.
Immunolabeling of Myocytes
When cardiac cells were labeled with the anti-titin antibody
T12, a single stripe appeared in the I band in close proximity to the Z
line (Fig 2). The distance between the center of the T12
epitope and the center of the Z line in slack sarcomeres was 100 nm.
The same distance was maintained in sarcomeres that had shortened
before immunolabeling to below the slack length, some of which were
shorter than the A-band width (Fig 2C). In sarcomeres that had been
stretched, the T12 epitope was also found 100 nm from the Z line
(Fig 2D and 2E). For example, at a sarcomere length of 2.0 µm, the
distance from the Z line to the epitope was 98.1±9.4 nm (n=9), and at
2.5 µm, the distance was 105±7 nm (n=13).
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Under the experimental conditions used in this study, the antibody
Ti-102 labeled a fine line at the edge of the A band (Fig 3). The distance between the Ti-102 epitope and the
middle of the A band was 841±40 nm (n=9) at a sarcomere length of 2.0
µm and 807±33 nm (n=13) at a length of 2.5 µm. The Ti-102 epitope
is thus at the very edge of the A band, and its position with respect
to the A band is independent of sarcomere length.
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Double labeling with both Ti-102 and T12 antibodies revealed that the
I-band segment of titin between the Ti-102 and T12 epitopes is elastic
(Fig 3C and 3D). The T12–to–Ti-102 segment is very short in
sarcomeres near the slack length (bottom sarcomeres of Fig 3D) and
highly elongated when sarcomeres are stretched (top sarcomeres of Fig 3D). At a length of 2.9 µm, both the T12 and Ti-102 epitopes lost
their alignment in the sarcomere, and the epitope zones broadened and
became fuzzy (Fig 4). Similar phenomena have been
reported for highly stretched skeletal muscle fibers6 19
and have been ascribed to detachment of the titin filament from the
thick filament. Our results in Fig 4 indicate that such detachment may
also occur in cardiac muscle. Additionally, misalignment of thick
filaments at high degrees of sarcomere stretch can also be expected to
result in broadening of the epitope zones.
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The anti-titin antibody 9D10 labeled a single epitope in the I
band, approximately in the middle of the segment demarcated by T12 and
Ti-102 (Fig 5). The 9D10 epitope position was sensitive
to sarcomere length and moved away from both the T12 and Ti-102
epitopes when sarcomeres were stretched (Fig 5). This indicates that
the titin segments between T12 and 9D10 (segment A) and the segment
between 9D10 and Ti-102 (segment B) are both elastic. We measured the
length of segment A and segment B at sarcomere lengths of 2.0 and 2.5
µm, respectively. Segment A increased in length from 37±11 (n=10) to
142±20 nm (n=11), whereas segment B increased from 64.3±21 (n=10) to
170±26 nm (n=11). In sarcomeres that were close to the slack length,
9D10, T12, and Ti-102 epitopes could not be distinguished separately,
because the epitopes were merged together (Fig 5C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We studied the length of the domain of the titin molecule in cardiac muscle that is elastic and mechanically active in developing passive tension. Specific locations along the titin molecule were marked by monoclonal antibodies, and their positions were followed by immunoelectron microscopy after stretching the sarcomere to varying lengths. Only a relatively small fraction (25% to 50%) of the unstrained I-band domain of titin turned out to be elastic.
The T12 epitope is located 100 nm from the center of the Z line, and
the Ti-102 epitope is located at the edge of the A band (Figs 2 and 3).
It is likely that the titin molecule extends beyond these epitopes,
because rat cardiac titin has been reported to cross-react with
anti-titin antibodies that in skeletal muscle label either in the A
band or in the region between the middle of the Z line and the T12
epitope (Reference 44 and K. Trombitás, unpublished data, 1995).
Thus, it is likely that in cardiac muscle a single titin molecule spans
from the Z line to the M line, as it does in skeletal muscle. The
finding that both the distance from the Z line to T12 and the distance
from Ti 102 to the M line are independent of sarcomere length (Figs 2 and 3) suggests that the 100-nm-long titin domain between the Z
line and the T12 epitope and the 800-nm-long domain between the
Ti-102 epitope and the M line are inelastic. Earlier, we estimated the
contour length of unstretched rat cardiac titin to be 1000
nm,7 and subtracting the inelastic domains leaves 100
nm to span from the T12 to the Ti-102 epitopes.
In skeletal muscle, T12 labels titin 100 nm from the Z line, and the
distance from the Z line to T12 is independent of sarcomere
length,4 13 as we found for cardiac muscle. Further, those
antibodies that label skeletal muscle either in the I band near the
edge of the A band or in the A band maintain their positions relative
to the M line over a wide range of sarcomere
lengths.13 19 20 Therefore, it is likely that the
100-nm-long titin domain that starts at the Z line and the
800-nm-long titin domain in the A band are inelastic in all
striated muscles.
It has been reported that the titin domain between the Z line and T12
epitope and the domain in the A band behave elastically in skeletal
muscle stretched to extreme lengths. At sarcomere lengths of >4
µm, epitopes close to the edge of the A band move into the I
band,6 12 whereas the T12 epitope moves away from the Z
line.13 We found the same in cardiac myocytes stretched to
lengths of >2.9 µm (Fig 4). In those highly extended sarcomeres,
the stress exerted on the T12 and Ti-102 epitopes by the elastic titin
domain exceeds the force with which these epitopes are anchored,
pulling them loose and allowing them to behave elastically. Thus, the
inelastic titin domains are intrinsically elastic but are normally
rendered inextensible by being anchored to other sarcomere structures.
The A-band domain of titin may be anchored to the thick
filament.1 2 3 Using cloned titin fragments,
Jin16 recently reported binding between titin and F-actin,
and this interaction may underlie the inelastic behavior of titin in
the I band. The finding that only the first 100 nm of the I-band domain
is inelastic suggests that the thin-filament region close to the Z
line is unique. A similar conclusion was drawn in a study on
tropomyosin localization in muscle.21
The 100-nm-long titin segment spanning from the T12 to the
Ti-102 epitopes is elastic, as indicated by the translocation of the
9D10 epitope away from both the T12 and Ti-102 epitopes that occurs
upon sarcomere stretch (Fig 5). It cannot be excluded, however, that
the T12–to–Ti-102 segment contains regions that are inelastic. It has
been shown in skeletal muscle that the I-band domain of titin contains
a 50-nm-long segment adjoining the A band that is
inelastic.6 If such a region existed in cardiac muscle,
then the remaining elastic domain would be only 50 nm long. Such
domain would be able to elongate maximally about
eightfold,22 allowing it to reach a length of 400 nm.
However, we found that the T12 to Ti-102 segment can attain a length of
550 nm before the T12 and Ti-102 epitopes start to translocate (Fig 4 and K. Trombitás, H. Granzier, unpublished data, 1995). If we
assume that this 550-nm-long segment has been strained
eightfold,22 then its unstrained length will be 70 nm.
Although the exact length of the unstrained extensible titin segment
remains to be established, it is likely to be somewhere between 50 and
100 nm, or 25% to 50% of the total I-band domain of titin. This is
much shorter than the unstrained extensible I-band domain of titin in
skeletal muscle. For example, in rabbit semitendinosus muscle, the
total I-band domain when unstrained is 350 nm,12 of
which 150 nm is inextensible4 6 13 and 200 nm is
extensible. For a given degree of sarcomere stretch, the extensible
I-band domain of titin will thus be strained to a much higher degree in
cardiac than in skeletal muscle, and this is likely to underlie the
much higher passive tensions developed by cardiac muscle (Fig 1).
The T12, 9D10, and Ti-102 epitopes all merge in sarcomeres that are
slack (Fig 5C). Therefore, it is likely that in slack sarcomeres the
elastic titin domain is folded on top of itself. This highly folded
state is probably similar to that of the nodules that are often seen
when purified native titin is viewed in the electron
microscope23 24 and to that of the highly retracted titin
seen in sarcomeres that have been
"freeze-broken."25 The entropy of such highly
folded titin will be maximal, and force will have to be exerted to
unfold it and lower its entropy.26 We propose that in
cardiac muscle the titin-based passive tension at sarcomere lengths
from 1.85 µm to 2.00 µm is developed by unfolding of the
elastic titin domain. At a sarcomere length of 2.0 µm, the I-band
width will be equal to the contour length of the total I-band domain of
titin,7 and the elastic titin domain will be fully
unfolded at this length. The domain will respond to further stretch by
elongating. Elongation continues until a sarcomere length of 2.9
µm is reached (Fig 4). At this length, the degree of elongation will
be maximal (eightfold extension), and further sarcomere stretch
results in recruitment of the inelastic A-band and Z-line domains to
the elastic pool. We also propose that during shortening to below the
slack length, titin develops a restoring force that brings sarcomeres
back to the slack length. In sarcomeres below the slack length, the T12
epitope remains 100 nm away from the Z line (Fig 2C). The ends of
the thick filaments will thus move past the T12 epitope toward the Z
line, extending the highly folded elastic titin domain in the process,
just as occurs when the sarcomere length increases beyond slack.
Unfolding of titin, both below and above the slack length, and titin
elongation at lengths longer than 2.0 µm occur over the working
range of sarcomeres in the heart (1.8 to 2.4 µm27 )
and are likely to play important roles in normal heart function.
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Acknowledgments |
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This study was supported by a Grant-in-Aid from the American Heart Association, Washington State Affiliate, Inc, to Dr Granzier, by a Whitaker Foundation grant for biomedical research to Dr Granzier, by a grant-in-aid from the Heart and Stroke Foundation of Alberta to Dr Jin, by a development grant from the Medical Research Council of Canada to Dr Jin, and by a Hungarian OTKA T6280 grant to Dr Trombitás. We express our gratitude to Dr Miklós Kellermayer for critical reading of various drafts of the manuscript and to Bronislava Stockman for superb technical assistance.
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Footnotes |
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Reprint requests to Dr Henk Granzier, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164-6520. E-mail granzier@unicorn.it.wsu.edu.
Received May 19, 1995; accepted August 1, 1995.
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 S. C. Lieber, N. Aubry, J. Pain, G. Diaz, S.-J. Kim, and S. F. Vatner Aging increases stiffness of cardiac myocytes measured by atomic force microscopy nanoindentation Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H645 - H651. [Abstract] [Full Text] [PDF]
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 M. M. LeWinter Titin Isoforms in Heart Failure: Are There Benefits to Supersizing? Circulation, July 13, 2004; 110(2): 109 - 111. [Full Text] [PDF]
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 H. L. Granzier and S. Labeit The Giant Protein Titin: A Major Player in Myocardial Mechanics, Signaling, and Disease Circ. Res., February 20, 2004; 94(3): 284 - 295. [Abstract] [Full Text] [PDF]
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 N. Fukuda, Y. Wu, G. Farman, T. C Irving, and H. Granzier Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle J. Physiol., November 15, 2003; 553(1): 147 - 154. [Abstract] [Full Text] [PDF]
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 M. Helmes, C. C. Lim, R. Liao, A. Bharti, L. Cui, and D. B. Sawyer Titin Determines the Frank-Starling Relation in Early Diastole J. Gen. Physiol., February 3, 2003; 121(2): 97 - 110. [Abstract] [Full Text] [PDF]
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W.-S. Lee, W.-P. Huang, W.-C. Yu, K.-R. Chiou, P. Y.-A. Ding, and C.-H. Chen Estimation of preload recruitable stroke work relationship by a single-beat technique in humans Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H744 - H750. [Abstract] [Full Text] [PDF]
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H. Granzier and S. Labeit Cardiac titin: an adjustable multi-functional spring J. Physiol., June 1, 2002; 541(2): 335 - 342. [Abstract] [Full Text] [PDF]
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T. S. Harris, C. F. Baicu, C. H. Conrad, M. Koide, J. M. Buckley, M. Barnes, G. Cooper IV, and M. R. Zile Constitutive properties of hypertrophied myocardium: cellular contribution to changes in myocardial stiffness Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2173 - H2182. [Abstract] [Full Text] [PDF]
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K. Watanabe, P. Nair, D. Labeit, M. S. Z. Kellermayer, M. Greaser, S. Labeit, and H. Granzier Molecular Mechanics of Cardiac Titin's PEVK and N2B Spring Elements J. Biol. Chem., March 22, 2002; 277(13): 11549 - 11558. [Abstract] [Full Text] [PDF]
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 L. Grama, B. Somogyi, and M. S. Z. Kellermayer Global configuration of single titin molecules observed through chain-associated rhodamine dimers PNAS, November 15, 2001; (2001) 191494098. [Abstract] [Full Text] [PDF]
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K. Trombitas, 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, October 1, 2001; 281(4): H1793 - H1799. [Abstract] [Full Text] [PDF]
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 M.-L. Bang, R. E. Mudry, A. S. McElhinny, K. Trombitas, A. J. Geach, R. Yamasaki, H. Sorimachi, H. Granzier, C. C. Gregorio, and S. Labeit Myopalladin, a Novel 145-Kilodalton Sarcomeric Protein with Multiple Roles in Z-Disc and I-Band Protein Assemblies J. Cell Biol., April 16, 2001; 153(2): 413 - 428. [Abstract] [Full Text] [PDF]
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 S. Hein, S. Kostin, A. Heling, Y. Maeno, and J. Schaper The role of the cytoskeleton in heart failure Cardiovasc Res, January 14, 2000; 45(2): 273 - 278. [Abstract] [Full Text] [PDF]
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O. Cazorla, A. Freiburg, M. Helmes, T. Centner, M. McNabb, Y. Wu, K. Trombitas, S. Labeit, and H. Granzier Differential Expression of Cardiac Titin Isoforms and Modulation of Cellular Stiffness Circ. Res., January 7, 2000; 86(1): 59 - 67. [Abstract] [Full Text] [PDF]
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M. Helmes, K. Trombitas, T. Centner, M. Kellermayer, S. Labeit, W. A. Linke, and H. Granzier Mechanically Driven Contour-Length Adjustment in Rat Cardiac Titin's Unique N2B Sequence : Titin Is an Adjustable Spring Circ. Res., June 11, 1999; 84(11): 1339 - 1352. [Abstract] [Full Text] [PDF]
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C. C. Gregorio, K. Trombitas, T. Centner, B. Kolmerer, G. Stier, K. Kunke, K. Suzuki, F. Obermayr, B. Herrmann, H. Granzier, et al. The NH2 Terminus of Titin Spans the Z-Disc: Its Interaction with a Novel 19-kD Ligand (T-cap) Is Required for Sarcomeric Integrity J. Cell Biol., November 16, 1998; 143(4): 1013 - 1027. [Abstract] [Full Text] [PDF]
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M. R. Zile, M. K. Cowles, J. M. Buckley, K. Richardson, B. A. Cowles, C. F. Baicu, G. Cooper IV, and V. Gharpuray Gel stretch method: a new method to measure constitutive properties of cardiac muscle cells Am J Physiol Heart Circ Physiol, June 1, 1998; 274(6): H2188 - H2202. [Abstract] [Full Text] [PDF]
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K. Trombitas, M. Greaser, S. Labeit, J.-P. Jin, M. Kellermayer, M. Helmes, and H. Granzier Titin Extensibility In Situ: Entropic Elasticity of Permanently Folded and Permanently Unfolded Molecular Segments J. Cell Biol., February 23, 1998; 140(4): 853 - 859. [Abstract] [Full Text] [PDF]
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S. Labeit, B. Kolmerer, and W. A. Linke The Giant Protein Titin: Emerging Roles in Physiology and Pathophysiology Circ. Res., February 1, 1997; 80(2): 290 - 294. [Abstract] [Full Text]
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M. Helmes, K. Trombitas, and H. Granzier Titin Develops Restoring Force in Rat Cardiac Myocytes Circ. Res., September 1, 1996; 79(3): 619 - 626. [Abstract] [Full Text]
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L. Grama, B. Somogyi, and M. S. Z. Kellermayer Global configuration of single titin molecules observed through chain-associated rhodamine dimers PNAS, December 4, 2001; 98(25): 14362 - 14367. [Abstract] [Full Text] [PDF]
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O. Cazorla, Y. Wu, T. C. Irving, and H. Granzier Titin-Based Modulation of Calcium Sensitivity of Active Tension in Mouse Skinned Cardiac Myocytes Circ. Res., May 25, 2001; 88(10): 1028 - 1035. [Abstract] [Full Text] [PDF]
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M.-L. Bang, T. Centner, F. Fornoff, A. J. Geach, M. Gotthardt, M. McNabb, C. C. Witt, D. Labeit, C. C. Gregorio, H. Granzier, et al. The Complete Gene Sequence of Titin, Expression of an Unusual {approx}700-kDa Titin Isoform, and Its Interaction With Obscurin Identify a Novel Z-Line to I-Band Linking System Circ. Res., November 23, 2001; 89(11): 1065 - 1072. [Abstract] [Full Text] [PDF]
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