Journal of Biomechanics 152 (2023) 111553

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Journal of Biomechanics
journal homepage: www.elsevier.com/locate/jbiomech

Review

Stretching the story of titin and muscle function

Wolfgang A. Linke a, b, c, 1
a
Institute of Physiology II, University of Münster, Germany
b
Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Germany
c
German Centre for Cardiovascular Research, Berlin, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: The discovery of the giant protein titin, also known as connectin, dates almost half a century back. In this review,
Muscle mechanics I recapitulate major advances in the discovery of the titin filaments and the recognition of their properties and
Skeletal muscle function until today. I briefly discuss how our understanding of the layout and interactions of titin in muscle
Sarcomere
sarcomeres has evolved and review key facts about the titin sequence at the gene (TTN) and protein levels. I also
Passive tension
touch upon properties of titin important for the stability of the contractile units and the assembly and mainte­
Elasticity
nance of sarcomeric proteins. The greater part of my discussion centers around the mechanical function of titin in
skeletal muscle. I cover milestones of research on titin’s role in stretch-dependent passive tension development,
recollect the reasons behind the enormous elastic diversity of titin, and provide an update on the molecular
mechanisms of titin elasticity, details of which are emerging even now. I reflect on current knowledge of how
muscle fibers behave mechanically if titin stiffness is removed and how titin stiffness can be dynamically
regulated, such as by posttranslational modifications or calcium binding. Finally, I highlight novel and exciting,
but still controversially discussed, insight into the role titin plays in active tension development, such as length-
dependent activation and contraction from longer muscle lengths.

1. Introduction muscle. However, I will also provide ample information on where we

stand today in our understanding of titin with regard to the mechanism
The contractile unit of muscle is the sarcomere, a complex protein of elasticity, the role titin plays in sarcomere stabilization and mainte­
structure of nearly crystalline order. The sarcomere has long been (and nance, how titin stiffness can be regulated, and how the mechanical
still is in some textbooks) portrayed simply as an array of two inter­ properties of titin affect active muscle contraction. Along the way, I will
digitating filament networks which slide relative to one another to discuss consequences for muscle structure and function when titin or its
produce muscle contraction: the myosin-based thick filaments and the sub-segments are deleted or when the titin springs are acutely cleaved.
actin-based thin filaments. Such a simple structure would be inherently Although I will touch upon aspects of titin properties in heart muscle, my
unstable, especially when force production commences at longer muscle focus will be on the mechanical function of titin in skeletal muscle. The
lengths where the overlap of actin and myosin filaments in the sarco­ important topic of titin as a target of disease-causing mutations in
mere is reduced. Luckily, nature has designed a third sarcomeric fila­ muscle (Savarese et al., 2016) falls outside the scope of my review. I
ment system, which is anchored at the Z-disk and also binds to the apologize that due to space restrictions, many relevant findings cannot
myosin filaments, in-between leaving a “free” part that is extensible and be covered here.
viscoelastic. The field of muscle contraction has come a long way in
understanding the properties and functions of this elastic filament
known today as titin. For this special issue of the Journal of Biomechanics 1.1. The discovery of elastic filaments in muscle
celebrating the 50 years anniversary of the International Society of
Biomechanics, I was asked to recapitulate the discovery of titin and the On a typical transmission electron micrograph of a sarcomere, the
evolution of its functional role. Thus, my review will summarize elastic (titin) filaments cannot be identified (Fig. 1A). However, first
important findings in the history of research on elastic filaments in evidence for their presence dates back to 1954, when it was reported
that the extraction of actin and myosin did not cause the sarcomeres to

E-mail address: wlinke@uni-muenster.de.

1
Institute of Physiology II, University of Münster, Germany, Robert-Koch-Str. 27B, 48149, Münster.

https://doi.org/10.1016/j.jbiomech.2023.111553
Accepted 14 March 2023
Available online 23 March 2023
0021-9290/© 2023 Elsevier Ltd. All rights reserved.
W.A. Linke Journal of Biomechanics 152 (2023) 111553

fall apart; they were still held together by a “ghost-like” set of remaining the Z-line and the thick filaments (Locker and Daines, 1980; Sjöstrand,
filaments (Huxley and Hanson, 1954). Somewhat later, thin longitudinal 1962); T-filaments or core filaments, linking Z-lines external to thick
filaments were shown to span the “gap” between Z-lines and thick fila­ filaments (McNeill and Hoyle, 1967) or through a thick-filament core
ments in sarcomeres stretched beyond overlap (Carlsen et al., 1961; (Guba et al., 1968), respectively; and connecting C-filaments, (again)
Huxley and Peachey, 1961; Sjöstrand, 1962). Unfortunately, firm con­ linking Z-lines to thick filaments (Garamvölgyi, 1966; Trombitas and
clusions about the layout and function of these filaments could not be Tigyi-Sebes, 1974; White and Thorson, 1973). The concept of elastic
reached, because of the interpretational limits of the electron micro­ filaments in sarcomeres only reached a new quality when biochemical
graphs. The nebulous existence of the filaments was then met with a evidence for their existence was provided.
confusing array of names given to them: S-filaments, linking Z-lines The first description of a major new myofibrillar protein was by
through the M− line (Hanson and Huxley, 1956); gap filaments, bridging Maruyama (1976), who called it connectin, and then by Wang et al.

Fig. 1. Three-filament sarcomere and molecular architecture of titin. A) Electron micrograph of a stretched skeletal muscle sarcomere. Scale bar, 0.5 µm. B)
Coomassie-stained, loose “titin” gel loaded with a mix of rabbit soleus and psoas muscle tissue (Neagoe et al., 2003). C) Simple cartoon around 1995 showing the titin
filament (red) in the sarcomere. Modified from Linke et al., 1996. D) Layout of the thick, thin and titin filaments within the sarcomere as understood today, and
banding pattern including Z-disk, I-band, A-band, and M-band. E) Domain composition of the human titin protein inferred from the complete meta-transcript (363
exons; 35,991 amino acids), which does not include exon 48 coding for the Novex-3 region as this contains a termination signal. “Exon No.“ refers to the exon rank in
the human titin gene (TTN; 364 exons). Ig, immunoglobulin-like; PEVK, proline, glutamic acid, valine and lysine rich region; FN3, fibronectin-type-3-like; TK, titin
kinase. F) Exon composition of titin isoforms expressed in skeletal muscles. N2A titin variants exist in very many different-length isoforms produced by alternative
splicing of I-band exons. Color indicates which type of protein domain an exon encodes.

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W.A. Linke Journal of Biomechanics 152 (2023) 111553

(1979), who called it titin — the name mostly used today. The protein which together with the demonstration of titin-myosin interactions
was initially estimated to have a molecular mass of ~ 1.4–2.8 MDa (Houmeida et al., 1995; Murayama et al., 1989) suggested that A-band
(Kurzban and Wang, 1988; Maruyama et al., 1984; Wang, 1982), titin may be a molecular ruler for the thick-filament length (Gregorio
whereas on loose protein gels used today (Fig. 1B), skeletal muscle titin et al., 1999; Trinick, 1996). This hypothesis has been controversially
runs between 3.3 and 3.7 MDa (Neagoe et al., 2003) — making it the discussed (Granzier et al., 2014; Myhre and Pilgrim, 2014; Tskhovre­
largest protein in our body. In the mid-1980 s, titin molecules were bova et al., 2015) but has been validated by genetic deletion of a few
isolated and found to have a string-like appearance on electron micro­ large super-repeats in the mouse, which resulted in correspondingly
graphs (Maruyama et al., 1984; Trinick et al., 1984; Wang et al., 1984) shorter A-bands (Tonino et al., 2017). Importantly, the mechanical
and a length of ~ 1 μm (Nave et al., 1989). Interactions of isolated titin stability of the A-band titin domains appears to be critical for their
with both actin and myosin were observed (Kimura et al., 1984). proper interaction with myosin (Rees et al., 2021). Titin’s large super-
Moreover, electron microscopical mapping of antibodies against repeat length also coincides with the distance between C-zone stripes
different Z-disk, I-band, A-band and M− band titin epitopes in muscle related to the presence of MyBPC (Freiburg and Gautel, 1996), although
tissues revealed that a titin molecule spans the half-sarcomere from the it is still disputed whether the MyBPC stripes and the titin super-repeats
Z-line (N-terminus) to the M− line (C-terminus) (Fürst et al., 1988; Itoh exactly match (Bennett et al., 2020; Tonino et al., 2019). Taken together,
et al., 1988; Maruyama et al., 1985; Wang et al., 1984; Whiting et al., A-band titin is a molecular blueprint for the sarcomeric A-band (uni­
1989). I-band titin is stretchable, whereas A-band titin is functionally formly 1.6 µm long in vertebrates!) and the arrangement of the myosin
stiff (Fürst et al., 1988; Higuchi et al., 1992; Itoh et al., 1988; Trombitas heads. This way, titin ensures proper A-band assembly (Fig. 2).
et al., 1991; Wang et al., 1991), due to interactions with myosin and
myosin-binding protein-C (MyBPC) (Fürst et al., 1989; Houmeida et al., 1.3. The titin kinase (TK) domain
1995; Labeit et al., 1992). Within the M− band, titin associates with
M− protein or myomesin (Obermann et al., 1996; Obermann et al., Near the A-band/M− band junction, titin contains a kinase domain
1997); for a recent review, see Lange et al. (2020). These and related (Labeit et al., 1992), which was initially suggested to be active during
findings established the layout of titin in the sarcomere (Fig. 1C, D). muscle development (Mayans et al., 1998) and involved in mechano-
Titin’s existence was confirmed beyond doubt when Labeit, Trinick chemical signal transduction (Puchner et al., 2008). However, current
and coworkers provided cDNA-sequence data, first for the A-band region understanding is that the TK-domain is rather a pseudokinase (Bogo­
of human heart and rabbit psoas titin (Labeit et al., 1990; Labeit et al., molovas et al., 2014) that functions as a scaffold supporting mechano­
1992) and, ultimately, for full-length human skeletal and cardiac titin sensing and proteostasis mechanisms (Fig. 2) (Bogomolovas et al., 2021;
(Labeit and Kolmerer, 1995). Titin has since been known as a single Bogomolovas et al., 2014; Lange et al., 2005). Specifically, the TK-
gene-encoded protein (gene name, TTN), which after myosin and actin is domain and a nearby upstream titin region bind muscle RING-finger
the third most abundant protein of vertebrate striated muscle, consti­ protein-1 and -2 (MuRF1/2) (Bogomolovas et al., 2021; Bogomolovas
tuting ~ 10–15 % of the total muscle protein pool. The inferred, com­ et al., 2014; Centner et al., 2001; Lange et al., 2005; McElhinny et al.,
plete meta-transcript of human titin (363 exons) predicts a theoretical 2002; Witt et al., 2005), which are E3-ligases that ubiquitinate various
protein length of 35,991 amino acids (NCBI reference number, myofilament proteins but especially distal A-band proteins, including
NP_001254479.2) (Fig. 1E, F). titin (Bogomolovas et al., 2021; Fomin et al., 2021; Müller et al., 2021;
Witt et al., 2005). Moreover, the TK-domain links titin to a macro­
autophagy pathway by interacting with Nbr1 in complex with
1.2. Titin as a molecular ruler for the sarcomeric A-band
sequestosome-1 (SQSTM1/p62) and MuRFs (Bogomolovas et al., 2021;
Lange et al., 2005). Thus, the TK-region is probably involved in coor­
An important finding of the sequencing studies was that A-band titin
dinating the targeted degradation of titin and associated proteins via the
is mainly organized in super-repeats (Labeit et al., 1990; Labeit et al.,
ubiquitin–proteasome system and the autophagy-lysosomal pathway
1992). Specifically, six 7-domain (small) and eleven 11-domain (large)
(Fig. 2), e.g., during regular protein turnover/maintenance. If distal titin
super-repeats (Fig. 1E) are formed in the so-called D-zone and C-zone of
regions including the TK-domain and MuRF-binding sites are deleted in
the half-A-band, respectively, by a repeating order of immunoglobulin-
mice, the skeletal muscles atrophy and the animals die ~ 5 weeks after
like (Ig) and fibronectin type-3-like (FN3) domains made up of ~
birth (Peng et al., 2005).
80–100 amino acids each and folded into 7–8-stranded β-barrels
(Improta et al., 1996; Muhle-Goll et al., 1998; Politou et al., 1994). The
length of the large super-repeat matches the 43-nm distance of the
myosin heads in the C-zone (Fürst et al., 1989; Whiting et al., 1989),

Fig. 2. Recognition of the functional diversity of titin. Today’s knowledge of titin properties includes the protein‘s multiple interactions with other sarcomeric
and non-sarcomeric proteins and the diverse functions as a sarcomeric template and stabilizer, mechanosensor, signaling hub, target of proteostasis mechanisms, and
substrate for Ca2+ binding and posttranslational modifications (such as phosphorylation or oxidation) regulating titin-based stiffness. Note that by far not all known
interactions of titin are displayed, only those discussed in this review (for details, see main text).

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W.A. Linke Journal of Biomechanics 152 (2023) 111553

1.4. Anchorage of elastic titin in the Z-disk tandem and are connected by short linkers providing some flexibility
and extensibility (Improta et al., 1996), like a “carpenter’s ruler” (von
Near its N-terminus, titin contains up to seven “Z-repeats” consisting Castelmur et al., 2008). The name-giving region of the N2A (and N2BA)
of 45 amino-acid long repeating motifs, which interact with α-actinin titin isoforms, termed N2-A element, is located in-between the differ­
(Gautel et al., 1996; Joseph et al., 2001; Ohtsuka et al., 1997; Sorimachi entially spliced Ig-domains and the PEVK-segment and is composed of
et al., 1997). This major Z-disk protein also crosslinks the barbed ends of four Ig-domains plus intervening sequences (Labeit and Kolmerer,
the actin filaments within the so-called Z-unit (Fig. 2). The titin Z-re­ 1995). As discussed later, the N2-A element is a mechanical signaling
peats are differentially spliced, which regulates the number of α-actinin- hub (Fig. 2).
titin crosslinks. The number of Z-repeats was suggested to determine the A few titin exons were overlooked in the early cDNA-sequencing
width of the Z-disk (Gautel et al., 1996; Young et al., 1998); however, studies but were added later (Bang et al., 2001). The most prominent
this is probably incorrect (Luther and Squire, 2002). Newer work has of these “novel” exons is Novex-3 (TTN-exon 48), which contains a
provided a refined atomic structure of α-actinin and its ligands (Gautel termination signal generating a short, Z-disk-anchored, titin isoform of
and Djinovic-Carugo, 2016; Ribeiro Ede et al., 2014). Force measure­ ~ 650 kDa also called Novex-3 (Fig. 1F). This isoform (expressed in
ments with optical tweezers revealed the mechanical stability of the skeletal and cardiac muscles) does not reach the A-band and probably is
α-actinin-titin bonds, the increase in bond strength with the number of not elastic. Novex-3 is involved in protein–protein interactions but
crosslinks, and the dynamic nature of these interactions (Grison et al., otherwise, is little characterized (Kellermayer et al., 2017). Modern
2017). next-generation sequencing approaches have since been useful to the
Various other interactions involving titin’s Z-disk region and their field, to validate the canonical sequence of titin and its modifications
functional implications have been demonstrated (reviewed by Frank and (Genomes Project., 2010).
Frey (2011); Knöll et al. (2011); Lange et al. (2006); Linke (2008);
Luther (2009); Solis and Solaro (2021); Wadmore et al. (2021)). A well- 1.6. The Cronos isoform of titin
established interaction occurs between the first two Ig-domains of two
titin filaments entering the Z-disk from the same half-sarcomere and T- Yet another muscle titin species was discovered much later, the
cap/telethonin (Fig. 2) (Gregorio et al., 1998; Mues et al., 1998). Cronos isoform (Zou et al., 2015). Cronos is under the control of an
Notably, this interaction does not provide the strong Z-disk anchorage of internal, alternative promoter located upstream of exon 241 in human
titin (Knöll et al., 2011) — the α-actinin–Z-repeat bonds do. However, TTN (Fig. 1F). Interestingly, Cronos is nearly absent in slow skeletal
there are mechanically relevant titin-actin interactions at the periphery muscles but prominently expressed in fast muscles (Swist et al., 2020;
of the Z-disk (Linke et al., 1997; Trombitas and Granzier, 1997) Zou et al., 2015). In adult human hearts, Cronos makes up ~ 12 % of all
involving the region coded by TTN-exon 28 (Linke et al., 1997). If the titin proteins (Fomin et al., 2021), whereas embryonic cardiomyocytes
actin filaments are experimentally extracted from the sarcomere, this express much more Cronos (Zaunbrecher et al., 2019). Like A-band titin,
part of titin becomes elastic (Linke et al., 1997; Trombitas and Granzier, Cronos may be a molecular ruler for the thick filaments as it is able to
1997). The network of titin-binding partners within the Z-disk is still support de-novo sarcomerogenesis in embryonic cardiomyocytes even
expanding (Filomena et al., 2021; Rudolph et al., 2020), also reflecting in the absence of full-length titins (Zaunbrecher et al., 2019). However,
the participation of Z-disk titin in multiple signaling pathways. In Cronos cannot maintain the sarcomere structure in adult mouse skeletal
summary, N-terminal titin is an integral part of the Z-disk that has both muscles depleted of the Z-disk-anchored titin isoforms (Swist et al.,
structural and signaling functions. 2020). The function of Cronos is still incompletely understood.

1.5. I-band titin architecture and composition 1.7. Diversity in titin elasticity and titin-based force

The cDNA sequencing (Labeit and Kolmerer, 1995) was also instru­ Arguably the best-known property of titin is the elasticity and ability
mental in showing that elastic titin undergoes extensive alternative to generate a “passive” force when stretched, as already proposed at the
splicing of exons (Fig. 1E, F) resulting in different-length titin isoforms. time of its discovery (Maruyama, 1976). Evidence obtained during the
In skeletal muscle, these isoforms are coined “N2A” (Fig. 1F), whereas mid-1980 s to mid-1990 s confirmed that titin must be responsible for at
the heart has “N2B” and “N2BA” (Freiburg et al., 2000), the latter of least part of the developed passive tension when nonactivated skeletal
which was initially called “N2A” as well (Labeit and Kolmerer, 1995). muscle is stretched (Funatsu et al., 1990; Granzier and Wang, 1993;
Both N2BA and N2A (but not N2B) exist in many lengths variants Magid and Law, 1985; Wang et al., 1991, 1993) and for restoring the
because of the I-band splicing diversity. Human N2A titins express 312 sarcomeres to their initial length after a physiological stretch (Trombitas
exons or less, out of the 364 exons present in TTN (Fig. 1F). It was et al., 1993; Wang et al., 1991). Even before the titin sequence was
important news that I-band titin contains up to ~ 100 Ig-domains, no published, different muscles were found to have a different titin protein
FN3-domain, and one or two large unique sequences (Labeit and Kol­ size, which is inversely correlated with the magnitude of passive tension
merer, 1995). One of them is expressed only in cardiac titin, the N2-B- the muscle develops (Horowits, 1992; Wang et al., 1991). When I
unique sequence (N2-Bus; ~570 residues), which is part of the N2-B worked as a postdoc in the laboratory of Prof. Gerald Pollack in the early
element coded by TTN-exon 49 (Fig. 1E). The other, even larger 1990 s, I learned how to measure the forces generated by a single
unique sequence (up to ~ 2,200 residues) is the PEVK-segment named so myofibril (Fig. 3). It turned out that an isolated cardiac myofibril is
after its high content of proline, glutamic acid, valine and lysine resi­ usually stiffer than an isolated skeletal myofibril (Bartoo et al., 1993;
dues. The PEVK-segment is an intrinsically disordered region organized Linke et al., 1994). We proposed that cardiac myofibrils — probably
as repeating motifs of mostly 28 amino acids, each typically coded by a their titin filaments — are responsible for most of the passive tension
single exon. PEVK encompasses as many as 114 exons in humans (TTN- even in multicellular cardiac specimens that contain extracellular matrix
exons 112–225), although some never seem to be expressed in muscle proteins (Linke et al., 1994). Later we reported that single myofibrils of
(Fig. 1F). Only the PEVK-exons 220–225 are constitutively expressed, all different skeletal muscle types also differ in their passive sarcomere
others are differentially spliced. length (SL)-tension relationships: sarcomeres of slow-type muscles (e.g.,
The Ig-domain regions of I-band titin were classified into constitu­ soleus) are typically less stiff than those of fast-type muscles (e.g.,
tively expressed “proximal” and “distal” segments, complemented by a psoas), which in turn are less stiff than cardiac sarcomeres (Fig. 3) (Linke
differentially spliced “middle” Ig-segment (Fig. 1E, F) (Freiburg et al., et al., 1998a; Linke et al., 1996; Linke et al., 1999). The primary
2000; Labeit and Kolmerer, 1995). An I-band Ig-domain is almost always sequence of titin (Labeit and Kolmerer, 1995) explained this elastic di­
encoded by a single TTN exon. These Ig-domains are usually arranged in versity, demonstrating that I-band titin is longer in soleus than in psoas

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W.A. Linke Journal of Biomechanics 152 (2023) 111553

sequences between the I-band Ig-domains were suggested to provide

extensibility (Politou et al., 1995). With the full titin sequence at hand
(Fig. 4A), the PEVK-domain also became an interesting candidate and
initially was proposed to be a more compliant spring than the Ig-
segments (Labeit and Kolmerer, 1995).
These hypotheses became testable when antibodies specific to I-band
titin segments were available. By immunostaining isolated myofibrils
(Fig. 4B) (Linke et al., 1998a; Linke et al., 1996) or muscle tissue (Gautel
and Goulding, 1996; Linke et al., 1998b; Trombitas et al., 1998), evi­
dence for the non-homogeneous extension of I-band titin segments was
obtained and a new “sequential extension” model put forth: at low
stretch forces, the linker sequences between Ig-domains extend, but
when their extensibility is (nearly) exhausted, higher stretch forces
extend the PEVK-segment (Fig. 4C). Indeed, reversible extension of
PEVK could readily be observed by immunofluorescence in stretched
Fig. 3. Diversity in titin-based passive tension among muscle types. Pas­
single myofibrils (Fig. 4B) (Linke et al., 1998a). Importantly, the
sive sarcomere-length tension relationships recorded on passively stretched
single myofibrils from different striated muscle types (rat). Quasi steady-state
sequential extension of Ig- and PEVK-segments occurs at physiological
“elastic” tension is shown. Inset: Single myofibril suspended for mechanical forces and SLs (Fig. 4C). A twist is that some muscle fibers co-express
measurements; scale bar, 3 µm. Modified from Neagoe et al., 2003. two titin isoforms (e.g., rabbit psoas; Fig. 1B; Prado et al. (2005)),
which might co-extend in the sarcomere (Fig. 4C). While the new
muscle, whereas cardiac N2B has the shortest I-band titin. The differ­ sequential titin-extension model (Gautel and Goulding, 1996; Linke
ences are brought about by splicing-in more or less middle Ig-domains et al., 1996) was quickly confirmed (Linke et al., 1998a; Linke et al.,
and PEVK-repeats. For details, I refer to relevant original articles and 1998b; Trombitas et al., 1998; Tskhovrebova and Trinick, 1997), it
reviews published at that time (Freiburg et al., 2000; Granzier et al., remained unclear for some time whether the Ig-domains of I-band titin
2000; Linke, 2000; Neagoe et al., 2003). unfold during physiological stretching.
The great elastic diversity of titin and its relevance to sarcomere Much of this discussion was based on the findings of three landmark
stiffness has since been revealed in many studies. For instance, each papers published back-to-back in 1997, which measured the force-
skeletal muscle may have its “personalized” titin-isoform length and extension relationships of isolated titin molecules or recombinant titin
titin-based stiffness (Li et al., 2012; Prado et al., 2005; Tirrell et al., fragments using the atomic force microscope (AFM; Rief et al. (1997)) or
2012). However, titin isoform-based stiffness and extracellular-matrix optical tweezers (Kellermayer et al., 1997; Tskhovrebova et al., 1997).
stiffness can be inversely correlated to one another (Prado et al., These studies found that titin Ig-domains unfold under relatively high
2005). Furthermore, developing hearts and muscles switch their I-band forces (150–250 pN in AFM force-extension experiments; Fig. 4D) and
titin springs from long/compliant in fetal tissues to shorter/stiffer during concluded that these unfolding forces are probably too high to be
and after birth (Lahmers et al., 2004; Opitz et al., 2004; Ottenheijm physiological. Interestingly, the unfolded titin domains refolded under
et al., 2009; Warren et al., 2004). The cardiac titin-isoform composition forces of a few piconewtons. The outstanding relevance of these papers
also varies in the different chambers of the heart (Cazorla et al., 2000; is based on their recognition of titin as a polymer whose elasticity can
Neagoe et al., 2003) and among different adult species, in that short/stiff largely be described as that of an entropic spring. Moreover, they firmly
titins predominate in small rodent hearts and longer/softer titins in large established the new field of single-molecule protein mechanics. These
animals (Cazorla et al., 2000; Neagoe et al., 2003). Moreover, the N2BA: experiments were also complemented with mathematical modelling,
N2B titin-isoform ratio is affected by hormones like insulin or thyroid especially steered molecular dynamics simulations, providing atomic-
hormone (Krüger et al., 2010; Krüger et al., 2008) and can be altered in level detail of domain unfolding events (comprehensively reviewed by
heart disease (Makarenko et al., 2004; Nagueh et al., 2004; Neagoe Hsin et al. (2011)). Since this review is not the place to recapitulate the
et al., 2002; Wu et al., 2002). A key factor regulating titin splicing is various important implications of these studies, I refer to relevant re­
RNA-binding motif 20 (RBM20; Guo et al. (2012)). This splicing factor views (Fisher et al., 2000; Granzier et al., 2002; Kellermayer and Grama,
regulates > 50 different proteins in the heart, including titin, but it also 2002; Linke and Fernandez, 2002; Linke and Grützner, 2008). Collec­
splices titin in skeletal muscles (Buck et al., 2014). Normal RBM20 ac­ tively, these and subsequent single-molecule experiments on titin (e.g.,
tivity promotes the shorter/stiffer titins, such as N2B in the heart, but Li et al. (2002); Marszalek et al. (1999); Nagy et al. (2005); Oberhauser
mutations in RBM20 (or deletion) repress I-band splicing, a giant titin et al. (2001); Watanabe et al. (2002)) validated the sequential titin-
(>3.8 MDa) remains (Guo et al., 2012), and heart disease can develop extension hypothesis (Fig. 4C), established and extended the concept
(Koelemen et al., 2021). In conclusion, titin’s elastic diversity suggests of titin as a molecular spring whose elasticity is entropically driven but
that titin-based stiffness is individually fine-tuned to allow the proper also has enthalpic components — e.g., entropic elasticity cannot explain
mechanical function of the different striated muscle types in diverse viscoelastic behavior but Ig-domain unfolding can (Minajeva et al.,
situations throughout the lifespan. 2001) — and thus, answered crucial questions about the molecular
mechanisms of titin elasticity.
The issue of “physiological” titin domain unfolding was re-addressed
1.8. Exploring the molecular mechanisms of titin elasticity much later, when a low-drift magnetic tweezers setup was used to hold
Ig-domain constructs under a very low, constant force (e.g., 5 pN) over
In the early 1990 s, A-band titin was suggested to be recruited to the time (Fig. 4E). Under these “physiological” stretch conditions, repetitive
elastic titin segment during higher physiological excursions, when the lengthening-shortening steps of ~ 10 nm were observed, clearly indi­
titin-myosin bonds at the thick-filament tips become disrupted (“yield cating Ig-domain unfolding-refolding transitions (Fig. 4E) (Rivas-Pardo
point”; (Wang et al., 1991, 1993)). However, the yield point occurs at ~ et al., 2016). Similar events with step sizes of ~ 10–13 nm appeared in
3.8–4.5 µm SL, depending on muscle type, which is outside of the immunolabeled single psoas myofibrils after a quick stretch to ~ 3.1 µm
physiological SL range (Linke et al., 1996). Other models of titin SL, corresponding to ~ 6 pN/titin (Rivas-Pardo et al., 2016). Because
extensibility assumed that the 7–8 stranded β-sheets of the Ig/FN3- shortening against a force means work production, Ig-domain refolding
domains can unfold (Pfuhl and Pastore, 1995; Soteriou et al., 1993), was proposed to be an “extra kick” in the power output of shortening
explaining titin elasticity (Erickson, 1994). In addition, the short linker muscle (Martonfalvi et al., 2017; Rivas-Pardo et al., 2016). While such a

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Fig. 4. Molecular mechanisms underlying titin elasticity. A) Details of the titin layout and architecture in the sarcomere, with realistic number of titin domains.
B) Example of anti-titin immunofluorescence labeling of a single myofibril stretched to different sarcomere lengths (in µm). Titin AB1 and 2 are antibodies to either
end of the PEVK segment. Scale bar, 3 µm. Modified from Linke et al., 1998a. C) Sequential extension model of structurally distinct elastic titin segments over a
physiological sarcomere length range (in µm). The cartoon depicts two different-length titin isoforms co-expressed in a muscle fiber (e.g., in rabbit psoas). Titin
domain numbers are not realistic. D) Unfolding of titin Ig domains in a recombinant fragment stretched in force-extension experiments using the atomic force
microscope (AFM). Modified from Li et al., 2002. E) Unfolding and refolding events measured in a recombinant titin Ig-domain construct stretched and held at a low,
constant force of 5 pN using a magnetic tweezers setup. Modified from Rivas-Pardo et al., 2016. F) Current understanding of how elastic titin stretches in the muscle
sarcomere and develops passive tension. Yield point: non-physiological stretch state causing the usually inextensible A-band titin at the thick filament tips to come
loose of myosin and be recruited to elastic titin.

scenario is well-founded and fascinating from a biophysical point of treatment of muscle samples (trypsin or high salt concentrations), in
view (Eckels et al., 2019), its physiological meaning in muscle remains order to dislodge the titin filaments from their anchorage points and
obscure (Linke, 2018). Our current understanding of the molecular thus eliminate titin-based stiffness (e.g., Granzier and Wang (1993);
mechanisms of titin elasticity is summarized in Fig. 4F, incorporating Hettige et al. (2022)). A potentially useful alternative, the genetic
sequential Ig-segment-PEVK extension, as well as the SL-dependent shift deletion of whole titin (in animal models) is not compatible with life
in Ig-domain unfolding and refolding probabilities (Fig. 4C). (Radke et al., 2019; Swist et al., 2020). If titin production is stopped in
the skeletal muscles of adult mice, other thick filament/M− band pro­
1.9. Contribution of titin to total muscle stiffness teins are lost (but not the Z-disk/thin filament proteins!), the sarcomeres
disintegrate, and the fibers lose their mechanical strength (Swist et al.,
Accumulating evidence has suggested that titin-based force is 2020). In mouse models with a partial deletion of I-band titin, the fibers
important for the passive force of muscle fibers. However, muscles generally demonstrate increased titin-based stiffness (Brynnel et al.,
consist of many protein structures potentially contributing to passive 2018; Buck et al., 2014; van der Pijl et al., 2020). While informative,
stiffness. The quantitation of titin’s contribution to stiffness is not titin deletion in animals often introduces secondary disease states,
straightforward and has often been based on unspecific chemical whose long-term consequences could mask the immediate mechanical

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effect of the titin modification on the sarcomeres. the extracellular matrix (Lieber and Binder-Markey, 2021). In our hands,
An elegant model to quantify titin stiffness is the titin-cleavage an additional observation in 100 % titin-cleaved and stretched muscle
mouse, in which a protease from the tobacco etch virus (TEV) can be fibers was the misalignment of Z-disks and A-bands (Fig. 5B, C), sug­
used to cleave I-band titin within a genetic cassette cloned into the distal gesting that titin stiffness — even if low, e.g., at 2.4 µm SL (Fig. 5B) — is
Ig-region (Rivas-Pardo et al., 2020). In homozygous mutant (Hom) mice, required to keep the sarcomeres organized and aligned during passive
100 % titin can be cleaved specifically and acutely by TEV, whereas titin stretching (Li et al., 2020). In summary, while the contribution of titin
is not cleaved by TEV in wildtype (Wt) mice. We employed this model to stiffness to total passive muscle stiffness requires further characteriza­
precisely measure the titin contribution to the SL-dependent passive tion, it is clear that titin is the main determinant of passive elastic (and
elastic force of permeabilized psoas muscle fiber bundles (Li et al., also viscous!) forces over the physiological SL range in fiber bundles.
2020). We found that below an SL of 3.2 µm (physiological range; Lle­
wellyn et al. (2008)), titin contributed 51–56 % (Fig. 5A). The remainder 1.10. Regulation of titin-based stiffness
was mainly attributed to the collagen fibers (still present in these sam­
ples), whose relative contribution to stiffness increased above ~ 3.2 µm A fascinating fact is that the stiffness of titin in muscle fibers is not
SL. It will be interesting to also use this mouse model to clarify the constant. As discussed, it depends on the titin-isoform pattern and I-
relevance of titin for the passive force of whole skeletal muscle in vivo, band spring composition. However, there are ways to also modulate titin
which has recently been claimed to be overwhelmingly determined by stiffness more dynamically and acutely. The best-studied of those
mechanisms involves posttranslational modifications (PTMs) of titin,
especially phosphorylation and oxidation (Beckendorf and Linke, 2015;
Linke and Hamdani, 2014). Much work has focused on cardiac titin,
where the N2-Bus element is a major site of phosphorylation and
oxidation (Loescher et al., 2022). Mechanistically, phosphorylation of
N2-Bus by a protein kinase (PK), e.g., PKA, PKG or calcium/calmodulin-
dependent kinase 2δ (CaMKIIδ), reduces titin-based stiffness (Hamdani
et al., 2013; Krüger et al., 2009; Yamasaki et al., 2002), and this can be
reversed by dephosphorylation via a protein phosphatase, such as PP5
(Krysiak et al., 2018). Deregulated cardiac titin phosphorylation is
associated with heart disease (Koser et al., 2019). Furthermore, oxida­
tive stress causes disulfide bonding within N2-Bus, which stiffens this
region (Grützner et al., 2009). However, titin can also be phosphory­
lated and oxidized in skeletal muscle. Hundreds of (potential) phos­
phosites and oxidizable cysteines are present in titin (Giganti et al.,
2018; Herrero-Galan et al., 2022; Koser et al., 2019; Loescher et al.,
2020), the functional relevance of which is largely unknown, especially
as regards the PTMs in A-band titin. A well-known substrate of PKs is the
constitutive PEVK-segment (Fig. 2), which can be phosphorylated by
PKCα (Hidalgo et al., 2009) or CaMKIIδ (Hamdani et al., 2013). The
transfer of (negatively charged) phosphate groups to this (positively
charged) PEVK sub-segment increases titin stiffness (Hidalgo et al.,
2009), probably by enhancing intramolecular electrostatic bonding.
Altered PEVK-phosphorylation as a potential cause of passive stiffness
changes has sometimes, but not consistently, been observed in diseased
or exercised muscle (Hidalgo et al., 2014; Müller et al., 2014; Otten­
heijm et al., 2012; Unger et al., 2017). Interestingly, unfolded Ig-
domains of titin can also be phosphorylated, although the functional
implications are unknown (Loescher et al., 2020). What is better un­
derstood are the mechanical consequences of unfolded (Ig) domain
oxidation (UnDOx) within elastic titin: S-glutathionylation of unfolded
domains reduces titin stiffness, whereas disulfide bonding increases it
(Alegre-Cebollada et al., 2014; Loescher et al., 2020). Other types of
PTMs, such as arginylation (Leite Fde et al., 2016) and acetylation
(Abdellatif et al., 2021), also affect titin stiffness, but the mechanisms
are unresolved.
Apart from PTMs, at least three other mechanisms alter titin-based
stiffness (Fig. 2). One is the binding of heat shock proteins (HSPs),
Fig. 5. Contribution of titin to passive tension and the stability of muscle especially small HSPs like αB-crystallin (CRYAB) and HSP27, to I-band
fibers. A) Passive sarcomere length (SL) - elastic force relationships of per­ titin (N2-Bus, N2-A, and Ig-domains; (Bullard et al., 2004; Kötter et al.,
meabilized psoas fiber bundles from the titin-cleavage mouse model. Wildtype 2014). In skeletal muscles under diverse stress conditions, including
(Wt) fibers are not cleaved by tobacco etch virus protease (TEV) and force re­ disease or intense exercise, CRYAB, HSP27, and the ATP-dependent
mains unaltered (left). In homozygous mutant fibers (Hom), 100 % titin chaperone HSP90, translocate from the cytosol to (unfolded) I-band
cleavage by TEV causes a drop in force at all SLs (right). Red numbers indicate
titin domains, probably to protect them from aggregation and quick
statistically significant decrease in mean force at a given SL (%). B) Electron
degradation (Fig. 2) (Kötter et al., 2014; Unger et al., 2017), while
micrographs of Wt (left) and Hom (right) muscle fibers following TEV treatment
and passive stretch experiments; scale bars, 1 µm. C) Interpretation of ultra­ slightly increasing titin-based passive stiffness (Bullard et al., 2004;
structural changes in passively stretched sarcomeres with intact titin (top, Wt) Unger et al., 2017). The binding of HSP90 to the N2-A element is
or after 100 % titin cleavage by TEV (bottom, Hom). Some elastic titin remains regulated by a co-chaperone, the methyltransferase Smyd2 (Donlin
bound to the thin filaments, most of it retracts to the Z-disk. Modified from Li et al., 2012). Another way to increase titin stiffness is through Ca2+-
et al., 2020. binding to the differentially spliced PEVK sub-segment, which has a net

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W.A. Linke Journal of Biomechanics 152 (2023) 111553

negative charge (Fig. 2) (Labeit et al., 2003). Although skeletal muscle

titins can express many PEVK-motifs, the mechanical effect of Ca2+-
binding is small (Joumaa et al., 2008; Labeit et al., 2003) and its
reversibility kinetics and functional relevance are unknown. Yet another
mechanism to modulate titin-based stiffness involves the established
binding of muscle ankyrin repeat protein (MARP) to the N2-A element
(Miller et al., 2003), which however includes actin as well, thus cross­
linking the titin spring with the actin filament (Fig. 2) (van der Pijl et al.,
2021; Zhou et al., 2021). MARPs exist in several variants (MARP1-3), of
which MARP1 (or CARP/Ankrd1) usually is present in trace amounts in
resting/healthy skeletal muscle but is upregulated in stressed/diseased
muscle (Swist et al., 2020; van der Pijl et al., 2019; Wette et al., 2021).
As a consequence, the increased formation of N2-A-MARP1-actin
crosslinks leads to relatively more extension of PEVK with sarcomere
stretch and enhanced titin-based stiffness (van der Pijl et al., 2021; Zhou
et al., 2021). The exciting implications of these findings for the me­
chanical properties of muscle have been discussed elsewhere (Hessel and
Linke, 2021). Moreover, the own actin-binding propensities of both the
N2-A element (Dutta et al., 2018) and skeletal muscle PEVK (Bianco
et al., 2007; Linke et al., 2002; Nagy et al., 2004), together with the
disease-relevant association of N2-A with the protease calpain-3 (Sor­
imachi et al., 1995), implicate this central I-band region as a mechanical
signaling hub (Nishikawa et al., 2020b).

1.11. Titin as a crucial factor in active muscle contraction

A long-standing topic only briefly discussed here in light of recent in-

depth reviews (Freundt and Linke, 2019; Herzog, 2018; Nishikawa,
2020), is the role titin plays in active contraction. In the late 1980 s,
ionizing radiation directed at a muscle to degrade titin (but also other
proteins) was found to lower active force, especially when the
contraction started at longer SLs (Horowits et al., 1986; Horowits and
Podolsky, 1988). It was concluded that intact titin keeps the thick fila­
ments in the center of the sarcomere during contraction at stretched
Fig. 6. Deficits in sarcomere ultrastructure arising during active contra­
lengths, ensuring optimal actin-myosin interactions. Recently, my group tion following specific cleavage of the titin springs. Homozygous mutant
has used the titin-cleavage mouse aiming to prove this property of titin, sarcomeres (length, ~2.7 µm) from the titin-cleavage mouse before treatment
while avoiding the unspecific effects of radiation damage (Li et al., with tobacco etch virus protease (Hom-TEV) and following 100 % titin cleavage
2020). We used TEV to specifically cleave 100 % titin in permeabilized, by TEV (Hom + TEV) in the passive sarcomere or when maximally activated in
stretched muscle fibers and then Ca2+-activated the muscle maximally Ca2+-containing buffer. Modified from Li et al., 2020.
(Fig. 6). We found that thick filament bundles or individual thick fila­
ments moved uncontrolledly toward the Z-disks during contraction, 2014; Rassier et al., 2005; Schappacher-Tilp et al., 2015). The molecular
which reduced active force. The damage to the sarcomeres progressed basis of the proposed acute titin stiffening is subject to an ongoing
with the duration of contraction, causing thick filaments to translocate debate but actin-titin interactions involving the PEVK-N2-A region and
through the Z-disk, misalign, and even shed myosin molecules from their possible effects of Ca2+ on these parameters are discussed.
tips, thus verifying and extending the findings of Horowits and Podolsky Finally, titin also plays a critical role in length-dependent activation
(1988). Damage tended to be higher when sarcomeres contracted at (LDA), which is characterized by increased contractile force after a
longer SLs. Another conclusion of our work was that the spring stiffness stretch of muscle (or heart; the molecular basis of the Frank-Starling
of intact titin in resting muscle is not sufficient to perform the A-band law). Initial evidence for titin as a factor in LDA of cardiac muscle was
centering function: it must increase considerably (~4-fold) during provided >20 years ago (Cazorla et al., 2001; Fukuda et al., 2001) and
activation (Li et al., 2020). Such an increase has indeed been predicted has since been extensively confirmed. However, LDA also occurs in
(Nishikawa, 2020) or measured before (Leonard and Herzog, 2010) and skeletal muscles and is, at least in part, titin-dependent (Hessel et al.,
additional evidence in support of elevated “non-crossbridge stiffness” 2022; Irving et al., 2011; Mateja et al., 2013). We have recently gained
has been provided in active muscle mechanics experiments (Herzog, insight into the underlying mechanisms of titin-dependent LDA by
2018; Powers et al., 2020; Rassier et al., 2015). The reasons behind the comparing thick and thin filament protein periodicities in small-angle X-
titin stiffness increase in stretched and activated muscle have not yet ray diffraction measurements on permeabilized muscle fibers that were
been identified, despite interesting candidate mechanisms, such as passively stretched before and after 50 % targeted titin cleavage (using
actin-titin interactions (Freundt and Linke, 2019; Herzog, 2018; Hessel the heterozygous titin-cleavage mouse; Hessel et al. (2022)). The acute
and Linke, 2021; Nishikawa, 2020; Tomalka et al., 2020). reduction in titin stiffness blunted the length-dependent priming of both
A related issue is the origin of residual force enhancement (RFE), a thin and thick filaments, as judged from characteristic changes in X-ray
phenomenon typically associated with eccentric muscle activity and reflection patterns. Moreover, active force measurements demonstrated
characterized by increased steady-state force following active muscle reduced myofilament Ca2+ sensitivity after 50 % titin cleavage (Hessel
stretching (Herzog, 2018). An impressive amount of work from the et al., 2022). We concluded that the stretched titin springs pulling on the
Herzog, Nishikawa, and other labs has suggested a titin-stiffening effect myosin filaments convert the myosin heads from “off” to “on”, priming
as the most probable explanation of RFE (e.g., Boldt et al., 2020; them for interaction with actin (Fig. 7). The concomitant priming of
Fukutani and Herzog, 2020; Herzog and Leonard, 2002; Leonard and actin and troponin requires the presence of non-crossbridge links
Herzog, 2010; Nishikawa, 2020; Nishikawa et al., 2020a; Powers et al.,

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W.A. Linke Journal of Biomechanics 152 (2023) 111553

Fig. 7. Length-dependent activation triggered by

a titin stretch. Titin-based elastic forces pulling on
the thick filament turn myosin heads from an “off-
state“ to an “on-state“, thus priming them for inter­
action with the thin filament. The thin filament
(actin, troponin) is also primed by the stretch,
perhaps made possible by non-myosin bridges
(MyBPC) connecting thick and thin filaments. The
model is based on structural data obtained by small-
angle X-ray diffraction measurements of 50 % titin-
cleaved vs non-cleaved, passively stretched mouse
psoas muscle fibers. Titin cleavage reverses thick and
thin filament priming and reduces the Ca2+ sensitivity
of the myofilaments. Modified from Hessel et al.,
2022.

between the thick and thin filaments in the passive state, possibly Beckendorf, L., Linke, W.A., 2015. Emerging importance of oxidative stress in regulating
striated muscle elasticity. J. Muscle Res. Cell Motil. 36, 25–36.
MyBPC. These findings strongly support a role for titin in LDA.
Bennett, P., Rees, M., Gautel, M., 2020. The axial alignment of titin on the muscle thick
In summary, titin has evolved from a “ghost-like” filament to a mere filament supports its role as a molecular ruler. J. Mol. Biol. 432, 4815–4829.
passive spring to an essential protein that determines the organization of Bianco, P., Nagy, A., Kengyel, A., Szatmari, D., Martonfalvi, Z., Huber, T.,
the sarcomere, adjusts its stiffness according to the functional re­ Kellermayer, M.S., 2007. Interaction forces between F-actin and titin PEVK domain
measured with optical tweezers. Biophys. J . 93, 2102–2109.
quirements of striated muscles, and tunes active contraction. Clearly, the Bogomolovas, J., Gasch, A., Simkovic, F., Rigden, D.J., Labeit, S., Mayans, O., 2014. Titin
nearly 50 year-long titin story has not yet come to an end. kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the
sarcomeric M-line. Open Biol. 4, 140041.
Bogomolovas, J., Fleming, J.R., Franke, B., Manso, B., Simon, B., Gasch, A.,
CRediT authorship contribution statement Markovic, M., Brunner, T., Knoll, R., Chen, J., Labeit, S., Scheffner, M., Peter, C.,
Mayans, O., 2021. Titin kinase ubiquitination aligns autophagy receptors with
Wolfgang A. Linke: Conceptualization, Funding acquisition, Inves­ mechanical signals in the sarcomere. EMBO Rep. 22, e48018.
Boldt, K., Han, S.W., Joumaa, V., Herzog, W., 2020. Residual and passive force
tigation, Project administration, Resources, Supervision, Writing – enhancement in skinned cardiac fibre bundles. J. Biomech. 109, 109953.
original draft, Writing – review & editing. Brynnel, A., Hernandez, Y., Kiss, B., Lindqvist, J., Adler, M., Kolb, J., van der Pijl, R.,
Gohlke, J., Strom, J., Smith, J., Ottenheijm, C., Granzier, H.L., 2018. Downsizing the
molecular spring of the giant protein titin reveals that skeletal muscle titin
Declaration of Competing Interest determines passive stiffness and drives longitudinal hypertrophy. Elife 7, e40532.
Buck, D., Smith 3rd, J.E., Chung, C.S., Ono, Y., Sorimachi, H., Labeit, S., Granzier, H.L.,
2014. Removal of immunoglobulin-like domains from titin’s spring segment alters
The author declares that he has no known competing financial in­ titin splicing in mouse skeletal muscle and causes myopathy. J. Gen. Physiol. 143,
terests or personal relationships that could have appeared to influence 215–230.
Bullard, B., Ferguson, C., Minajeva, A., Leake, M.C., Gautel, M., Labeit, D., Ding, L.,
the work reported in this paper.
Labeit, S., Horwitz, J., Leonard, K.R., Linke, W.A., 2004. Association of the
chaperone alphaB-crystallin with titin in heart muscle. J. Biol. Chem. 279,
Acknowledgements 7917–7924.
Carlsen, F., Knappeis, G.G., Buchthal, F., 1961. Ultrastructure of the resting and
contracted striated muscle fiber at different degrees of stretch. J. Biophys. Biochem.
I thank the past and present members of my laboratory involved in Cytol. 11, 95–117.
the studies described. My research is supported by the German Research Cazorla, O., Freiburg, A., Helmes, M., Centner, T., McNabb, M., Wu, Y., Trombitas, K.,
Labeit, S., Granzier, H., 2000. Differential expression of cardiac titin isoforms and
Foundation (SFB1002A08), IZKF Münster (Li1/029/20), the German
modulation of cellular stiffness. Circ. Res. 86, 59–67.
Center for Cardiovascular Research, and the European Union. Cazorla, O., Wu, Y., Irving, T.C., Granzier, H., 2001. Titin-based modulation of calcium
sensitivity of active tension in mouse skinned cardiac myocytes. Circ. Res. 88,
1028–1035.
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