© 2000 American Heart Association, Inc.
Molecular Medicine |
Series of Exon-Skipping Events in the Elastic Spring Region of Titin as the Structural Basis for Myofibrillar Elastic Diversity
From the European Molecular Biology Laboratory (A.F., T.C., B.K., S.L.), Heidelberg, Germany; Institut für Anästhesiologie und Operative Intensivmedizin (A.F., C.W.), Universitätsklinikum Mannheim, Germany; Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology (K.T., O.C., H.G.), Washington State University, Pullman, Wash; Medigenomix (W.H.), Martinsried, Germany; Genethon (F.F., J.B.), Evry, France; and the Department of Cell Biology and Anatomy (C.C.G.), University of Arizona, Tucson, Ariz.
Correspondence to Dr Siegfried Labeit, EMBL, Meyerhofstrasse 1, 60012 Heidelberg, Germany. E-mail labeit{at}embl-heidelberg.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.
Key Words: atrium • titin • elasticity • diastole
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In addition to the active muscle force, myofibrils also generate passive forces when stretched above or shortened to below the slack length. Passive forces are directed to restore thick and thin filament overlap at rest; ie, vertebrate myofibrils have an intrinsic elasticity that is independent from the active force generating crossbridges. This elastic force resides within a third filament system, which is formed by titin. The giant polypeptide chain of titin is
3 MDa in size, and in situ it spans from Z- to M-lines, a
distance of 1 to 2 µm (for reviews on titin elasticity, see
References 1 and 2 ). During
physiological amounts of myofibril stretch, 90%
of the elastic passive tension response of cardiac muscle is derived
from the titin filament system, whereas particularly in the heart
muscle, collagen and intermediate filaments become important when
preventing further nonphysiological
overstretch.3 Three different sequence elements account for the extensibility of the I-band region of titin. One spring element is composed of tandemly arranged Ig domains. This element extends 3- to 4-fold at low forces, presumably by unbending interdomain linkers, whereas the Ig domains themselves maintain their tertiary structures.4 5 A second spring element, referred to as the PEVK segment, is formed by a sequence region rich in proline (P), glutamine (E), valine (V), and lysine residues (K).6 The PEVK spring produces most of the physiological passive tension response of titin, presumably by functioning as an entropic spring. Finally, the so-called N2B sequence of cardiac titin extends toward the end of the physiological sarcomere length (SL) range. In situ, the tandem Ig, PEVK, and N2B segments act as a serially linked multiple-spring system with elements that are sequentially recruited during stretch.7 8 9
The elastic properties of myofibrils from different vertebrate species and tissues are highly divergent, reflecting functional specialization. Tissue-specific expression of titin isoforms with different elasticities was suggested by gel-electrophoretic and sequencing studies.6 10 11 12 However, details of how muscles express highly diverse titins are unclear. Here, we determined the human gene sequence of titin that codes for its elastic I-band region. Characterization of titin transcripts from cardiac and skeletal muscles shows that cascades of exon-skipping events produce titins with distinct I-band structures and that myogenic differentiation results in muscle types with unique titin-based elastic properties.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Sequence Data
The 106-kb human titin gene sequence spanning from I24 to I84 is available from the European Molecular Biology Laboratory data library (EMBL), accession No. AJ277892; the rabbit titin soleus and cardiac N2B cDNA sequences are under accession Nos. Y14852 and Y14853.
Isoform Transcript Studies
RNAs from different rabbit muscles were analyzed by
reverse transcriptase–polymerase chain reaction (RT-PCR) with
combinations of I15S-to-I84R primers. cDNA clones were isolated from
human adult heart, human skeletal leg muscle, and rabbit skeletal psoas
muscle cDNA libraries with N2B/N2A probes. Human fetal embryos (age
from 4 to 7 postovulatory weeks) were from legal abortions induced by
mifepristone (RU486), approved by the Ethical Committee Necker Hospital
Paris. In situ hybridization probes were chimeric 30-meric antisense
oligonucleotides of which the 15 5' base pairs were
from the donor and the 15 3' bases were from the acceptor exon.
Antibodies, SDS-PAGE, and Western Blot Analysis of
Titin
For origin of the titin antibodies used and gel electrophoresis
and Western blot conditions, see online Materials and Methods
(http://www.circresaha.org). For epitope locations, see Figure 1
.
|
Passive Tension Measurements
Cardiac myocytes were from rat (male Sprague-Dawley, 250 g)
or pig (Yorkshire-type swine, 20 to 30 kg). For myofibril attachment
and passive tension measurements, see online Materials and Methods
(http://www.circresaha.org).
Immunoelectron and Immunofluorescence Microscopy
Skeletal muscle fibers and cardiac cells were stretched, fixed,
immunolabeled, embedded, and processed for electron microscopy, as
described online (http://www.circresaha.org). Indirect
immunofluorescence microscopy of 8- to 10-µm
frozen sections of rat (n=5) and rabbit (n=2) ventricular
and atrial myocardium and pig ventricular
myocardium (n=2) was essentially as
described.8
An expanded Materials and Methods section is available online at http://www.circresaha.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A 106-kb Genomic Region Codes for the Differentially Expressed Spring Families of Titin
Two sequenced titin isoforms from human myocardium and human soleus skeletal muscle cDNA libraries, referred to as N2B and N2A titins, have homologous A-band but strikingly different I-band regions (Figure 1
).6 To investigate the molecular basis of
titin diversity, we determined the human gene sequence of titin within
the differing N2A/N2B/PEVK regions (for details, see online Materials
and Methods [http://www.circresaha.org]). Within the PEVK region, the
gene sequence indicates the presence of at least 27 novel exons
that are not included in the cardiac and skeletal titin cDNA sequences
(Figure 1A). Twenty-five of the novel PEVK exons are 80 to 89 bp
in length and highly homologous to each other and code for
27-residue PEVK elements (Figure 1B). Two new exons 5' of N2B
together code for 8 novel Ig repeats. Previously, we named Ig repeats
by counting them in human cardiac titin.6 The discovery of
novel titin I-band Ig repeats interferes with this terminology. Below,
we refer to individual I-band Ig repeats by counting them from 5' to 3'
(N to C terminally) in the gene sequence. On the basis of this system,
the differentially spliced region spans I15 to I84 and encompasses 161
exons (Figure 1). In the N2B heart isoform, 155 exons are
skipped by splicing together the I27 and PEVK exons, which are 101 kb
apart (Figure 1). The N2B segment is coded for by a single
2.7-kb exon containing repeats I24/I25/I26. The N2A segment containing
I80 to I83 is coded for by a group of 6 exons 35 kb farther downstream
(Figure 1A).
Different Exon-Skipping Pathways Produce N2A, N2B, and N2BA
Titins
To study the tissue-specific splicing of the titin gene in an
animal model, we amplified cDNAs from 12 different rabbit muscles with
I15S-to-I84R primer pairs. The N2A exons are expressed in all striated
muscles (Figure 2A). In skeletal
muscles, the N2B exon is excluded by splicing together I15/I27 (Figure 2A), whereas inclusion of N2B is obligatory in the adult heart
(Figure 2B). To further characterize N2B cardiac titin(s), I27S
together with I28R-to-I84R primers was tested for amplification of
rabbit cardiac cDNAs. I27S+I84R amplified a "minimal-size" N2B
titin isoform in which the Ig repeats I28 to I83 and 92% of the
N-terminal PEVK domain are excluded. Further cardiac isoforms were
amplified with I27S+I61R and I27S+I82R, which contain both the N2A and
N2B elements but differ by multiple skipping variants in the I27-to-I68
segment (Figures 2B and 3). The
exon skips within I27 to I68 varied in different anatomical regions of
the rabbit heart (Figure 2B). In human, multiple variants were
also identified in the I27-to-I68 segment when clones were selected by
conventional filter screens with I27-to-I84 probes. Extensions to the
3' end with I79S identified a 600-residue human PEVK domain isoform
that is derived from the full-length soleus PEVK by a complex series of
skipping events (Figure 3). Finally, RT-PCR analysis of
rabbit skeletal muscles showed that only soleus muscle expresses I27 to
I34 as a continuous fragment, whereas all other tested striated muscles
skip I30 to I34. For example, psoas muscle skips I30 to I47 (Figures 2A and 3). During development, in situ hybridizations of
human fetal sections with titin anti-sense
oligonucleotides directed to the N2B-to-I27 and
I27-to-I68 junctions show that these splice routes are specific to the
heart at week 10 (Figure 2C). Therefore, yet unknown factors
determine the tissue specificity of titin splice routes during early
development.
|
HindIII size marker; lane 1, heart left ventricle; lane
2, M. psoas; lane 3, M. longissimus dorsi; lane 4, M. soleus; lane 5,
M. gastrocnemius; lane 6, M. plantaris longus; lane 7, diaphragm; lane
8, M. extensor digitorum longus; lane 9, M. tibialis anterior; lane 10,
M. rectus femoris; lane 11, tongue; lane 12, M. pectoralis major.
Tissue-specific exon skips occur in the proximal tandem Ig segment. A
contiguous I27-to-I36 segment is expressed only in soleus muscle.
Skipping of I28 to I47, I28 to I54, and I28 to I67 occurs in psoas
(lane 2) and heart muscle (lane 1). A, Right, I80 to I83 (N2A segment)
is expressed in all striated muscles, whereas expression of I24 to I26
(N2B exon) is restricted to cardiac muscle. B, Left, PCR amplification
of total human cDNAs from fetal cardiac (hfh), adult cardiac (hh), and
skeletal adult (hskA and hskB) muscles with N2B flanking I15S+I27R
primers. Skeletal muscles join exons I15 to I27, whereas cardiac adult
muscle includes N2B exonic I24/I25/I26 between I15 and I27. In human
fetal heart (week 20), N2B including and excluding splice routes
coexists (hh-1, control template of a sequenced cardiac cDNA clone). B,
Right, Multiple N2B-based cardiac isoforms result from joining I27 to
acceptors in I28 to I68. Arrows/labels indicate the respective Ig
repeats joined by splicing. LV indicates left ventricle; RV, right
ventricle; LA, left atrium. C, In situ hybridization of fetal human
sections. Antisense probes complementary to I26/I27 and I27/I68
junctions (right) stain specifically the developing heart, whereas the
constitutive control probe from the M-line titin region also stains the
surrounding body wall muscles (week 10, left) and limb muscles (weeks
10 and 20).
|
In summary, skeletal titin transcripts are N2A based (ie, they
always include the N2A segment and exclude the N2B exon), whereas
cardiac titins are N2B based (ie, cardiac titins always include the N2B
exon). Furthermore, in heart, very different splice routes for titin
coexist. One major cardiac splice route joins the I15-to-I24/I27-to-I84
exons and therefore is predicted to express a small cardiac titin (N2B
titin with 2.97 MDa). A second pathway includes both the N2A and N2B
segments, plus 12 to 25 tandem Ig repeats (I55 to I79) and a
600-residue-long PEVK domain (cardiac N2BA titin with a predicted
3.3 MDa). In psoas and soleus skeletal muscles, distinct splice
pathways produce cDNAs coding for larger titin isoforms (3.4 MDa in
psoas and 3.7 MDa in soleus). All titin isoforms identified so far
share the I27 exon, and much of the isoform diversity of titin results
from its differential splicing to multiple downstream acceptor exons
within I28 to I84 (Figure 3). Interestingly, the intron 3' to
I27 shares extensive sequence homology with 2 introns from the
differentially spliced PEVK region, raising the possibility that
conserved intron motifs are implicated in exon skipping (Figure 3).
Differentially Processed Titin Transcripts Translate Into
Titin Isoforms
If many structurally different titins are expressed, this
complicates how proper stoichiometry and assembly into sarcomeres could
be achieved. Therefore, we tested whether titin splice isoforms
translate into distinct polypeptides. In agreement with earlier
studies, low-percentage SDS gradient gels detect differently sized
titins in soleus, psoas, and heart muscle (Figure 4A, see also References
10 11 12 ). Western blot analysis with
anti-I55/I56/I57, anti-N2A-to-I80/I81, and anti-N2B antibodies shows
that skeletal titins are N2A based (Figure 4B), whereas cardiac
titins are N2B based (Figure 4C). Different titin size classes
are detected in myocardium of some species.
Myocardium of all investigated species contains a major T1
band with the same mobility, and an additional lower-mobility band is
found in some of the investigated species. This additional band is
present at low levels in rabbit and high levels in pig and human,
whereas the band cannot be detected in rat myocardium
(Figure 4A, middle and right panels). The lower-mobility band
reacts with all tested antibodies (anti-I80/I81 [N2A], anti-N2B, and
anti-I55/I56/I57 antibodies), whereas the higher-mobility band reacts
only with anti-N2B antibodies (Figure 4C). These results suggest
that the lower-mobility band corresponds to an N2BA titin isoform,
whereas the higher-migrating band is an N2B titin. Comparison of
cardiac titins from ventricles and atria of different species suggests
the coexpression of variable amounts of N2B and N2BA titins (Figure 4D and Reference 13 ). Avian (chicken and
turkey) myocardium expresses a single titin band that has a
mobility similar to that of rat N2B titin, presumably
representing N2B titin as well. Ventricular
myocardium from dog and cow expresses high levels of a
low-mobility titin that most likely represent N2BA titin. When
comparing ventricular and atrial myocardium,
N2BA titin is more abundant in the atrial tissue (Figure 4D).
The 2 distinct cardiac size classes differentially reacting with
anti-I55/I56/I57 and with anti-I80/81-N2A antibodies (Figure 4)
is consistent with the coexpression of N2B and N2BA titins in
the heart (Figure 3). These 2 isoforms are expressed at
different levels in different species, and at different levels in
atrial and ventricular myocardium.
|
Immunostaining with anti-I55/56/57 antibodies
(recognizing the large N2BA titin isoform) detect this isoform in a
subset (which appear to be randomly distributed) of cardiac myocytes.
The frequency of expressing cells varied between species, ranging from
a few percent in rat (1±1.4%) (Figure 5A) and rabbit (2.8±3.1%)
(Figure 5C) to abundant I55/I56/I57 expression in pig
(91±9.7%) (Figure 5E) and human myocardium (not
shown). In immunoelectron microscopy, both the anti-I24/I25 (N2B) and
anti-I80/I81 (N2A) antibodies label close to the middle of the I-band
region of the sarcomere (Figures 5B-2 and 5B-3). The N2B epitope
was on average closer to the Z-line than the N2A epitope. For example,
at an SL of 2.4 µm, the epitope distance to the middle of the
Z-line was 153±12 nm (n=6) for the N2B epitope and 270±30 nm (n=7)
for the N2A epitope. When the tissue was double labeled with both N2B
and N2A antibodies, the labeling pattern varied and sarcomeres were
found with 2 epitopes (Figure 5B-4), as well as sarcomeres that
contained a single epitope only (Figure 5B-5). When a single
epitope was found, the distance to the Z-line suggested that it was
derived from the N2B antibody and that the N2A epitope was absent.
These results are consistent with the coexpression of
structurally different N2B and N2BA titins in human
myocardium.
|
I-Band Titin Isoform Type and Passive Force Generation
Cardiac myocytes were isolated from rat and pig
myocardium, as these species represent examples of
those that express high levels of N2B titin (rat) and high levels of
N2BA titin (pig). Results indicate that passive tension of rat myocytes
increases much more steeply with SL than that of pig myocytes (Figure 6A). Thus, the expression of N2B cardiac
titin is associated with a high passive tension–generating
ability.
|
The passive properties of muscle fibers isolated from rabbit soleus and
psoas muscle were also studied. In sarcomeres longer than 2.5
µm, passive tension was found to increase much more steeply with SL
in psoas than in soleus fibers (Figure 6B). To explore the
origin of this, the lengths of the tandem Ig and PEVK segments were
measured by immunoelectron microscopy (for examples of labeled
sarcomeres, see Figure 7A). The lengths
of these segments were measured in sarcomeres stretched to different
lengths, and the segment lengths were expressed as a fraction of their
contour length (for technical details see online Materials and Methods;
http://www.circresaha.org). Results indicate that the fractional
extension of tandem Ig and PEVK segments in sarcomeres shorter than
2.5 µm are similar in psoas and soleus fibers. However, at
longer SLs the fractional extensions increase more steeply in psoas
than in soleus fibers (Figures 7B and 7C). The significance of
these findings is discussed below.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The titin isoform family is encoded by a single gene located on chromosome 2, region 2q31, and its locus is part of a group of genes that maintained conserved syntenic order since the divergence of rodents and humans.14 This study indicates that extensive exon shuffling in the I-band region of titin generates its isoform diversity, which is in charge of myofibrillar elastic diversity. Some of the splice pathway choices occur early and irreversibly during myogenesis, such as the cardiac-specific inclusion of the N2B exon (Figure 2
). Other aspects of titin splice regulation, such as
the myocardial N2B/N2BA isoform ratio, appear to be controlled more
flexibly, because different amounts of N2B and N2BA titin isoforms are
found in different compartments of the heart (Reference
13 and the present study). From the large number
of titin splice isoforms in different muscle types suggested by RT-PCR
experiments, we focused on defining the titin splice routes in 3 muscle
types that have different elastic properties: skeletal soleus, psoas,
and heart muscle. The soleus muscle expresses the largest titin isoform
observed so far (3.7 MDa), whereas psoas muscle expresses a smaller
titin (3.35 MDa) with shorter proximal tandem Ig and PEVK segments. In
the heart, titin transcripts are processed by 2 distinct splice routes
that lead to N2B and N2BA titins (2.97 and 3.3 MDa, respectively).
Interestingly, the lengths of the tandem Ig and PEVK domains appear to
correlate in the isoforms, which appears to require a coordination of
their splicing within a 100-kb genomic segment. It remains to be seen
whether the conserved introns in the tandem Ig and the PEVK regions
participate in exon skip regulation and whether they provide a basis
for tandem-Ig/PEVK splice coordination. So far, individual Ig/FN3 repeats in titin were numbered on the basis of their location in the cardiac isoform.6 For the repeats in the extensively spliced I-band region, it is now difficult to use this numbering consistently. Moreover, the titin gene codes for additional Ig repeats of yet-unknown function and tissue expression. Therefore, we propose to identify Ig repeats in the I-band by counting them from 5' to 3' in the human gene sequence. This nomenclature would allow for the systematic naming of the numerous splice pathways on the basis of the repeats that are spliced together (ie, I27-to-I84 splice pathway for the small cardiac N2B isoform, and I27 to I55 for the large cardiac N2BA isoform).
Tissue-specific variation in passive mechanical properties has been reported in many previous studies. They concluded that passive cardiac myocytes are much stiffer than passive skeletal muscle fibers, and that among different skeletal muscles, psoas muscle fibers are relatively stiff.2 13 15 16 Our work extends these earlier findings. We found that passive tension increase is steeper with SL in psoas than in soleus fibers. Also, the passive tension increase is steeper in myocytes isolated from rat myocardium, which express predominantly the small N2B isoform, than in myocytes isolated from pig myocardium, which express high levels of the large N2BA isoform. The differences in tandem Ig and PEVK segment length of the different muscle types provide a molecular framework to understand these mechanical differences.
The effect of tandem Ig and PEVK segment lengths on passive force can
be evaluated by considering the molecular mechanism of passive force
generation. A passive model has emerged recently in which the tandem-Ig
segments (containing folded Ig domains) and the PEVK segment (acting
largely as an unfolded polypeptide) behave as serially linked entropic
springs.7 8 9 In short sarcomeres, these springs are in a
contracted state with high entropy. On sarcomere extension the springs
straighten, lowering their conformational entropy and resulting in a
force, known as entropic force. This force increases with the
fractional extension of the segment (end-to-end length divided by the
contour length). The serially linked entropic springs model of passive
force development may be applied to titin isoforms by adapting the
entropic forces to the fractional extensions multiplied by the contour
lengths of the tandem Ig and PEVK segments of the isoform. Contour
lengths of tandem Ig and PEVK segments of psoas titin are 100 and
400 nm shorter, respectively, than in soleus titin, assuming a 5-nm
repeat per Ig domain and 3.8 Å per PEVK residue. Thus, for a given SL
the fractional extension will be higher in psoas that in soleus muscle
(Figures 7B and 7C). It follows that entropic force is predicted
to be much higher in psoas than in soleus fibers, consistent
with the measured passive tension differences (Figure 6B). A
similar analysis can be applied to the cardiac isoforms. The
contour lengths of tandem Ig and PEVK segments are 100 and 200 nm
shorter, respectively, in N2B than in N2BA titin. Thus, at a given SL
the fractional extension of tandem Ig and PEVK segments is considerably
higher for N2B titin than for N2BA titin and, therefore, passive force
will be higher. This prediction is qualitatively unaffected by the N2B
sequence, which all cardiac titin isoforms share. Thus, the serially
linked entropic springs model predicts that the passive force–SL
relation increases more steeply for cells containing high levels of N2B
titin (rat) than for cells containing high levels of N2BA titin (pig).
Consistent with the predicted differences, rat myocytes
generate higher passive forces than pig myocytes (Figure 6A).
In conclusion, this work provides a molecular basis for understanding the diversity in passive mechanical properties of muscle. Plasticity in splicing in the I15-to-I84 segment results in isoforms that vary in contour length. As a result, the fractional extension–SL relation of different isoforms differs, and this gives rise to distinctly different passive force–SL relations. Thus, the differential expression of the spring region of titin presents a striking example of how titin elasticity is modulated by splicing. Possibly, splice plasticity may also allow functional adaptations of the titin springs in training or muscle disease. Future studies are warranted to identify the factors in charge of titin splice choices and how their action may be regulated by muscle function and pathology.
|
Acknowledgments |
|---|
We gratefully acknowledge the support of the Deutsche Forschungsgemeinschaft (Grant La 668/6-1 to S.L.), the American Heart Association (Grant 98-WA-115 to O.C.), the NIH (Grants HL61497 and HL62881 to H.G. and HL57461 and HLO3985 to C.C.G.), the AFM (to J.B. and F.F.), and the HFSP (to S.L. and C.C.G.). We thank M. Vekemans and T. Attie from the Hopital Necker-Enfants Malades for provision of the embryonic tissues and M. Greaser (Wisconsin University) for communication of results on the 28–amino acid residue repeats in the PEVK.17 This work is dedicated to the memory of Alexandra Freiburg (9/10/72-7/14/99). We all miss her.
|
Footnotes |
|---|
1 Deceased.
Received March 14, 2000; accepted April 17, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Erickson HP. Stretching single protein molecules: titin is a weird spring. Science. 1997;276:1090–1092.
2. Linke WA, Granzier H. A spring tale: new facts on titin elasticity. Biophys J. 1998;75:2613–2614.[Medline] [Order article via Infotrieve]
3. Granzier HL, Irving TC. Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments. Biophys J. 1995;68:1027–1044.[Medline] [Order article via Infotrieve]
4. Linke WA, Ivemeyer M, Olivieri N, Kolmerer B, Rüegg JC, Labeit S. Towards a molecular understanding of the elasticity of titin. J Mol Biol. 1996;261:62–71.[Medline] [Order article via Infotrieve]
5.
Trombitas K, Greaser M, Labeit S, Jin JP, Kellermayer
M, Helmes M, Granzier H. Titin extensibility in situ: entropic
elasticity of permanently folded and permanently unfolded molecular
segments. J Cell Biol. 1998;140:853–859.
6.
Labeit S, Kolmerer B. Titins, giant proteins in charge
of muscle ultrastructure and elasticity. Science. 1995;270:293–296.
7.
Linke WA, Ivemeyer M, Mundel P, Stockmeier MR,
Kolmerer B. Nature of PEVK-titin elasticity in skeletal muscle.
Proc Natl Acad Sci U S A. 1995;95:8052–8057.
8. Linke WA, Rudy DE, Centner T, Gautel M, Witt CC, Labeit S, Gregorio CC. I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure. J Cell Biol. 1999;246:631–644.
9.
Helmes M, Trombitas K, Centner T, Kellermayer M,
Labeit S, Linke WA, Granzier H. Mechanically driven contour-length
adjustment in rat cardiac titin’s unique N2B sequence: titin is an
adjustable spring. Circ Res. 1999;84:1339–1352.
10.
Hu DH, Kimura S, Maruyama K. Sodium dodecyl
sulfate gel electrophoresis studies of connectin-like high molecular
weight proteins of various types of vertebrate and invertebrate
muscles. J Biochem (Tokyo). 1986;99:1485–1492.
11.
Wang K, McCarter R, Wright J, Beverly J,
Ramirez-Mitchell R. Regulation of skeletal muscle stiffness and
elasticity by titin isoforms: a test of the segmental extension model
of resting tension. Proc Natl Acad Sci U S A. 1991;88:7101–7105.
12. Horowits R. Passive force generation and titin isoforms in mammalian skeletal muscle. Biophys J. 1992;61:392–398.[Medline] [Order article via Infotrieve]
13.
Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M,
Trombitas K, Labeit S, Granzier H. Differential expression of cardiac
titin isoforms and modulation of cellular stiffness. Circ
Res. 2000;86:59–67.
14. Rossi E, Faiella A, Zeviani M, Labeit S, Floridia G, Brunelli S, Cammarata M, Boncinelli E, Zuffardi O. Order of six loci at 2q24–q31 and orientation of the HOXD locus. Genomics. 1994;24:34–40.[Medline] [Order article via Infotrieve]
15.
Brady AJ. Length dependence of passive stiffness in
single cardiac myocytes. Am J Physiol. 1991;260:H1062–H1071.
16.
Fabiato A, Fabiato F. Myofilament-generated tension
oscillations during partial calcium activation and
activation dependence of the sarcomere length-tension relation of
skinned cardiac cells. J Gen Physiol. 1978;72:667–699.
17. Greaser ML, Wang S-M, Berri M, Mozdziak PE, Kumazawa Y. Sequence and mechanical implications of cardiac PEVK. In: Pollack GH, Granzier H, eds. Proceedings: Elastic Filaments of the Cell. Kluwer Academic/Plenum Publishers. In press.
This article has been cited by other articles:
 |
|
 |
|
  M. Kruger, S. Kotter, A. Grutzner, P. Lang, C. Andresen, M. M. Redfield, E. Butt, C. G. dos Remedios, and W. A. Linke Protein Kinase G Modulates Human Myocardial Passive Stiffness by Phosphorylation of the Titin Springs Circ. Res., January 2, 2009; 104(1): 87 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger Designing Heart Performance by Gene Transfer Physiol Rev, October 1, 2008; 88(4): 1567 - 1651. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Joumaa, D. E. Rassier, T. R. Leonard, and W. Herzog The origin of passive force enhancement in skeletal muscle Am J Physiol Cell Physiol, January 1, 2008; 294(1): C74 - C78. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Aartsma-Rus and G.-J. B. van Ommen Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications RNA, October 1, 2007; 13(10): 1609 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Granzier, M. Radke, J. Royal, Y. Wu, T. C. Irving, M. Gotthardt, and S. Labeit Functional genomics of chicken, mouse, and human titin supports splice diversity as an important mechanism for regulating biomechanics of striated muscle Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2007; 293(2): R557 - R567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Seeley, W. Huang, Z. Chen, W. O. Wolff, X. Lin, and X. Xu Depletion of Zebrafish Titin Reduces Cardiac Contractility by Disrupting the Assembly of Z-Discs and A-Bands Circ. Res., February 2, 2007; 100(2): 238 - 245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Olsson, M. Kruger, L.-H. Meyer, L. Ahnlund, L. Gransberg, W. A. Linke, and L. Larsson Fibre type-specific increase in passive muscle tension in spinal cord-injured subjects with spasticity J. Physiol., November 15, 2006; 577(1): 339 - 352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kruger, T. Kohl, and W. A. Linke Developmental changes in passive stiffness and myofilament Ca2+ sensitivity due to titin and troponin-I isoform switching are not critically triggered by birth Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H496 - H506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. C. Ottenheijm, L. M. A. Heunks, T. Hafmans, P. F. M. van der Ven, C. Benoist, H. Zhou, S. Labeit, H. L. Granzier, and P. N. R. Dekhuijzen Titin and Diaphragm Dysfunction in Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., March 1, 2006; 173(5): 527 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. G. Prado, I. Makarenko, C. Andresen, M. Kruger, C. A. Opitz, and W. A. Linke Isoform Diversity of Giant Proteins in Relation to Passive and Active Contractile Properties of Rabbit Skeletal Muscles J. Gen. Physiol., October 31, 2005; 126(5): 461 - 480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Huebsch, E. Kudryashova, C. M. Wooley, R. B. Sher, K. L. Seburn, M. J. Spencer, and G. A. Cox Mdm muscular dystrophy: interactions with calpain 3 and a novel functional role for titin's N2A domain Hum. Mol. Genet., October 1, 2005; 14(19): 2801 - 2811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Lim and D. B. Sawyer Modulation of Cardiac Function: Titin Springs into Action J. Gen. Physiol., February 28, 2005; 125(3): 249 - 252. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sarkar, S. Caamano, and J. M. Fernandez The Elasticity of Individual Titin PEVK Exons Measured by Single Molecule Atomic Force Microscopy J. Biol. Chem., February 25, 2005; 280(8): 6261 - 6264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Fujita, D. Labeit, B. Gerull, S. Labeit, and H. L. Granzier Titin isoform-dependent effect of calcium on passive myocardial tension Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2528 - H2534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Nagy, P. Cacciafesta, L. Grama, A. Kengyel, A. Malnasi-Csizmadia, and M. S. Z. Kellermayer Differential actin binding along the PEVK domain of skeletal muscle titin J. Cell Sci., November 15, 2004; 117(24): 5781 - 5789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Makarenko, C.A. Opitz, M.C. Leake, C. Neagoe, M. Kulke, J.K. Gwathmey, F. del Monte, R.J. Hajjar, and W.A. Linke Passive Stiffness Changes Caused by Upregulation of Compliant Titin Isoforms in Human Dilated Cardiomyopathy Hearts Circ. Res., October 1, 2004; 95(7): 708 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Borisov, A. Kontrogianni-Konstantopoulos, R. J. Bloch, M. V. Westfall, and M. W. Russell Dynamics of Obscurin Localization During Differentiation and Remodeling of Cardiac Myocytes: Obscurin as an Integrator of Myofibrillar Structure J. Histochem. Cytochem., September 1, 2004; 52(9): 1117 - 1127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Nagueh, G. Shah, Y. Wu, G. Torre-Amione, N. M.P. King, S. Lahmers, C. C. Witt, K. Becker, S. Labeit, and H. L. Granzier Altered Titin Expression, Myocardial Stiffness, and Left Ventricular Function in Patients With Dilated Cardiomyopathy Circulation, July 13, 2004; 110(2): 155 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Opitz, M. C. Leake, I. Makarenko, V. Benes, and W. A. Linke Developmentally Regulated Switching of Titin Size Alters Myofibrillar Stiffness in the Perinatal Heart Circ. Res., April 16, 2004; 94(7): 967 - 975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lahmers, Y. Wu, D. R. Call, S. Labeit, and H. Granzier Developmental Control of Titin Isoform Expression and Passive Stiffness in Fetal and Neonatal Myocardium Circ. Res., March 5, 2004; 94(4): 505 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Bullard, C. Ferguson, A. Minajeva, M. C. Leake, M. Gautel, D. Labeit, L. Ding, S. Labeit, J. Horwitz, K. R. Leonard, et al. Association of the Chaperone {alpha}B-crystallin with Titin in Heart Muscle J. Biol. Chem., February 27, 2004; 279(9): 7917 - 7924. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
G. Miller, H. Musa, M. Gautel, and M. Peckham A targeted deletion of the C-terminal end of titin, including the titin kinase domain, impairs myofibrillogenesis J. Cell Sci., December 1, 2003; 116(23): 4811 - 4819. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Opitz, M. Kulke, M. C. Leake, C. Neagoe, H. Hinssen, R. J. Hajjar, and W. A. Linke Damped elastic recoil of the titin spring in myofibrils of human myocardium PNAS, October 28, 2003; 100(22): 12688 - 12693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Warren, M. C. Jordan, K. P. Roos, P. R. Krzesinski, and M. L. Greaser Titin isoform expression in normal and hypertensive myocardium Cardiovasc Res, July 1, 2003; 59(1): 86 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Gentil, C. Delphin, C. Benaud, and J. Baudier Expression of the Giant Protein AHNAK (Desmoyokin) in Muscle and Lining Epithelial Cells J. Histochem. Cytochem., March 1, 2003; 51(3): 339 - 348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Huang, Q. Zhou, P. Liang, M. S. Hollander, F. Sheikh, X. Li, M. Greaser, G. D. Shelton, S. Evans, and J. Chen Characterization and in Vivo Functional Analysis of Splice Variants of Cypher J. Biol. Chem., February 21, 2003; 278(9): 7360 - 7365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Huq, E. K. Heist, and R. J. Hajjar Titin--Springing Back to Youth? Sci. Aging Knowl. Environ., December 11, 2002; 2002(49): pe20 - 20. [Abstract] [Full Text]
|
|
|
|
|
|
C. Neagoe, M. Kulke, F. del Monte, J. K. Gwathmey, P. P. de Tombe, R. J. Hajjar, and W. A. Linke Titin Isoform Switch in Ischemic Human Heart Disease Circulation, September 10, 2002; 106(11): 1333 - 1341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wu, S. P. Bell, K. Trombitas, C. C. Witt, S. Labeit, M. M. LeWinter, and H. Granzier Changes in Titin Isoform Expression in Pacing-Induced Cardiac Failure Give Rise to Increased Passive Muscle Stiffness Circulation, September 10, 2002; 106(11): 1384 - 1389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Rankinen, P. An, L. Perusse, T. Rice, Y. C. Chagnon, J. Gagnon, A. S. Leon, J. S. Skinner, J. H. Wilmore, D. C. Rao, et al. Genome-wide linkage scan for exercise stroke volume and cardiac output in the HERITAGE Family Study Physiol Genomics, August 14, 2002; 10(2): 57 - 62. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Lindstedt, T. E. Reich, P. Keim, and P. C. LaStayo Do muscles function as adaptable locomotor springs? J. Exp. Biol., August 1, 2002; 205(15): 2211 - 2216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Yamasaki, Y. Wu, M. McNabb, M. Greaser, S. Labeit, and H. Granzier Protein Kinase A Phosphorylates Titin's Cardiac-Specific N2B Domain and Reduces Passive Tension in Rat Cardiac Myocytes Circ. Res., June 14, 2002; 90(11): 1181 - 1188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 T. Toursel, L. Stevens, H. Granzier, and Y. Mounier Passive tension of rat skeletal soleus muscle fibers: effects of unloading conditions J Appl Physiol, April 1, 2002; 92(4): 1465 - 1472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 M. Kulke, C. Neagoe, B. Kolmerer, A. Minajeva, H. Hinssen, B. Bullard, and W. A. Linke Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle J. Cell Biol., September 3, 2001; 154(5): 1045 - 1058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V Person, S Kostin, K Suzuki, S Labeit, and J Schaper Antisense oligonucleotide experiments elucidate the essential role of titin in sarcomerogenesis in adult rat cardiomyocytes in long-term culture J. Cell Sci., January 11, 2000; 113(21): 3851 - 3859. [Abstract] [PDF]
|
|
|
|
|
|
G. Gutierrez-Cruz, A. H. Van Heerden, and K. Wang Modular Motif, Structural Folds and Affinity Profiles of the PEVK Segment of Human Fetal Skeletal Muscle Titin J. Biol. Chem., March 2, 2001; 276(10): 7442 - 7449. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
M. Kulke, S. Fujita-Becker, E. Rostkova, C. Neagoe, D. Labeit, D. J. Manstein, M. Gautel, and W. A. Linke Interaction Between PEVK-Titin and Actin Filaments: Origin of a Viscous Force Component in Cardiac Myofibrils Circ. Res., November 9, 2001; 89(10): 874 - 881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||