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

The contractile unit of muscle is the sarcomere, a complex protein
structure of nearly crystalline order. The sarcomere has long been (and
still is in some textbooks) portrayed simply as an array of two inter­
digitating filament networks which slide relative to one another to
produce muscle contraction: the myosin-based thick filaments and the
actin-based thin filaments. Such a simple structure would be inherently
unstable, especially when force production commences at longer muscle
lengths where the overlap of actin and myosin filaments in the sarco­
mere is reduced. Luckily, nature has designed a third sarcomeric fila­
ment system, which is anchored at the Z-disk and also binds to the
myosin filaments, in-between leaving a “free” part that is extensible and
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
celebrating the 50 years anniversary of the International Society of
Biomechanics, I was asked to recapitulate the discovery of titin and the
evolution of its functional role. Thus, my review will summarize
important findings in the history of research on elastic filaments in
stand today in our understanding of titin with regard to the mechanism
of elasticity, the role titin plays in sarcomere stabilization and mainte­
nance, how titin stiffness can be regulated, and how the mechanical
properties of titin affect active muscle contraction. Along the way, I will
discuss consequences for muscle structure and function when titin or its
sub-segments are deleted or when the titin springs are acutely cleaved.
Although I will touch upon aspects of titin properties in heart muscle, my
focus will be on the mechanical function of titin in skeletal muscle. The
important topic of titin as a target of disease-causing mutations in
muscle (Savarese et al., 2016) falls outside the scope of my review. I
apologize that due to space restrictions, many relevant findings cannot
be covered here.
1.1. The discovery of elastic filaments in muscle
On a typical transmission electron micrograph of a sarcomere, the
elastic (titin) filaments cannot be identified (Fig. 1A). However, first
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
filaments (Huxley and Hanson, 1954). Somewhat later, thin longitudinal
filaments were shown to span the “gap” between Z-lines and thick fila­
ments in sarcomeres stretched beyond overlap (Carlsen et al., 1961;
Huxley and Peachey, 1961; Sjöstrand, 1962). Unfortunately, firm con­
clusions about the layout and function of these filaments could not be
reached, because of the interpretational limits of the electron micro­
graphs. The nebulous existence of the filaments was then met with a
confusing array of names given to them: S-filaments, linking Z-lines
through the M− line (Hanson and Huxley, 1956); gap filaments, bridging
the Z-line and the thick filaments (Locker and Daines, 1980; Sjöstrand,
1962); T-filaments or core filaments, linking Z-lines external to thick
filaments (McNeill and Hoyle, 1967) or through a thick-filament core
(Guba et al., 1968), respectively; and connecting C-filaments, (again)
linking Z-lines to thick filaments (Garamvölgyi, 1966; Trombitas and
Tigyi-Sebes, 1974; White and Thorson, 1973). The concept of elastic
filaments in sarcomeres only reached a new quality when biochemical
evidence for their existence was provided.
The first description of a major new myofibrillar protein was by
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.

2
W.A. Linke
(1979), who called it titin — the name mostly used today. The protein
was initially estimated to have a molecular mass of ~ 1.4–2.8 MDa
(Kurzban and Wang, 1988; Maruyama et al., 1984; Wang, 1982),
whereas on loose protein gels used today (Fig. 1B), skeletal muscle titin
runs between 3.3 and 3.7 MDa (Neagoe et al., 2003) — making it the
largest protein in our body. In the mid-1980 s, titin molecules were
isolated and found to have a string-like appearance on electron micro­
graphs (Maruyama et al., 1984; Trinick et al., 1984; Wang et al., 1984)
and a length of ~ 1 μm (Nave et al., 1989). Interactions of isolated titin
with both actin and myosin were observed (Kimura et al., 1984).
Moreover, electron microscopical mapping of antibodies against
different Z-disk, I-band, A-band and M− band titin epitopes in muscle
tissues revealed that a titin molecule spans the half-sarcomere from the
Z-line (N-terminus) to the M− line (C-terminus) (Fürst et al., 1988; Itoh
et al., 1988; Maruyama et al., 1985; Wang et al., 1984; Whiting et al.,
1989). I-band titin is stretchable, whereas A-band titin is functionally
stiff (Fürst et al., 1988; Higuchi et al., 1992; Itoh et al., 1988; Trombitas
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.,
1995; Labeit et al., 1992). Within the M− band, titin associates with
M− protein or myomesin (Obermann et al., 1996; Obermann et al.,
1997); for a recent review, see Lange et al. (2020). These and related
findings established the layout of titin in the sarcomere (Fig. 1C, D).
Titin’s existence was confirmed beyond doubt when Labeit, Trinick
and coworkers provided cDNA-sequence data, first for the A-band region
of human heart and rabbit psoas titin (Labeit et al., 1990; Labeit et al.,
1992) and, ultimately, for full-length human skeletal and cardiac titin
(Labeit and Kolmerer, 1995). Titin has since been known as a single
gene-encoded protein (gene name, TTN), which after myosin and actin is
the third most abundant protein of vertebrate striated muscle, consti­
tuting ~ 10–15 % of the total muscle protein pool. The inferred, com­
plete meta-transcript of human titin (363 exons) predicts a theoretical
protein length of 35,991 amino acids (NCBI reference number,
NP_001254479.2) (Fig. 1E, F).
1.2. Titin as a molecular ruler for the sarcomeric A-band
An important finding of the sequencing studies was that A-band titin
is mainly organized in super-repeats (Labeit et al., 1990; Labeit et al.,
1992). Specifically, six 7-domain (small) and eleven 11-domain (large)
super-repeats (Fig. 1E) are formed in the so-called D-zone and C-zone of
the half-A-band, respectively, by a repeating order of immunoglobulin-
like (Ig) and fibronectin type-3-like (FN3) domains made up of ~
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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Journal of Biomechanics 152 (2023) 111553
which together with the demonstration of titin-myosin interactions
(Houmeida et al., 1995; Murayama et al., 1989) suggested that A-band
titin may be a molecular ruler for the thick-filament length (Gregorio
et al., 1999; Trinick, 1996). This hypothesis has been controversially
discussed (Granzier et al., 2014; Myhre and Pilgrim, 2014; Tskhovre­
bova et al., 2015) but has been validated by genetic deletion of a few
large super-repeats in the mouse, which resulted in correspondingly
shorter A-bands (Tonino et al., 2017). Importantly, the mechanical
stability of the A-band titin domains appears to be critical for their
proper interaction with myosin (Rees et al., 2021). Titin’s large super-
repeat length also coincides with the distance between C-zone stripes
related to the presence of MyBPC (Freiburg and Gautel, 1996), although
it is still disputed whether the MyBPC stripes and the titin super-repeats
exactly match (Bennett et al., 2020; Tonino et al., 2019). Taken together,
A-band titin is a molecular blueprint for the sarcomeric A-band (uni­
formly 1.6 µm long in vertebrates!) and the arrangement of the myosin
heads. This way, titin ensures proper A-band assembly (Fig. 2).
1.3. The titin kinase (TK) domain
Near the A-band/M− band junction, titin contains a kinase domain
(Labeit et al., 1992), which was initially suggested to be active during
muscle development (Mayans et al., 1998) and involved in mechano-
chemical signal transduction (Puchner et al., 2008). However, current
understanding is that the TK-domain is rather a pseudokinase (Bogo­
molovas et al., 2014) that functions as a scaffold supporting mechano­
sensing and proteostasis mechanisms (Fig. 2) (Bogomolovas et al., 2021;
Bogomolovas et al., 2014; Lange et al., 2005). Specifically, the TK-
domain and a nearby upstream titin region bind muscle RING-finger
protein-1 and -2 (MuRF1/2) (Bogomolovas et al., 2021; Bogomolovas
et al., 2014; Centner et al., 2001; Lange et al., 2005; McElhinny et al.,
2002; Witt et al., 2005), which are E3-ligases that ubiquitinate various
myofilament proteins but especially distal A-band proteins, including
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
sequestosome-1 (SQSTM1/p62) and MuRFs (Bogomolovas et al., 2021;
Lange et al., 2005). Thus, the TK-region is probably involved in coor­
dinating the targeted degradation of titin and associated proteins via the
ubiquitin–proteasome system and the autophagy-lysosomal pathway
(Fig. 2), e.g., during regular protein turnover/maintenance. If distal titin
regions including the TK-domain and MuRF-binding sites are deleted in
mice, the skeletal muscles atrophy and the animals die ~ 5 weeks after
birth (Peng et al., 2005).
W.A. Linke
1.4. Anchorage of elastic titin in the Z-disk
Near its N-terminus, titin contains up to seven “Z-repeats” consisting
of 45 amino-acid long repeating motifs, which interact with α-actinin
(Gautel et al., 1996; Joseph et al., 2001; Ohtsuka et al., 1997; Sorimachi
et al., 1997). This major Z-disk protein also crosslinks the barbed ends of
the actin filaments within the so-called Z-unit (Fig. 2). The titin Z-re­
peats are differentially spliced, which regulates the number of α-actinin-
titin crosslinks. The number of Z-repeats was suggested to determine the
width of the Z-disk (Gautel et al., 1996; Young et al., 1998); however,
this is probably incorrect (Luther and Squire, 2002). Newer work has
provided a refined atomic structure of α-actinin and its ligands (Gautel
and Djinovic-Carugo, 2016; Ribeiro Ede et al., 2014). Force measure­
ments with optical tweezers revealed the mechanical stability of the
α-actinin-titin bonds, the increase in bond strength with the number of
crosslinks, and the dynamic nature of these interactions (Grison et al.,
2017).
Various other interactions involving titin’s Z-disk region and their
functional implications have been demonstrated (reviewed by Frank and
Frey (2011); Knöll et al. (2011); Lange et al. (2006); Linke (2008);
Luther (2009); Solis and Solaro (2021); Wadmore et al. (2021)). A well-
established interaction occurs between the first two Ig-domains of two
titin filaments entering the Z-disk from the same half-sarcomere and T-
cap/telethonin (Fig. 2) (Gregorio et al., 1998; Mues et al., 1998).
Notably, this interaction does not provide the strong Z-disk anchorage of
titin (Knöll et al., 2011) — the α-actinin–Z-repeat bonds do. However,
there are mechanically relevant titin-actin interactions at the periphery
of the Z-disk (Linke et al., 1997; Trombitas and Granzier, 1997)
involving the region coded by TTN-exon 28 (Linke et al., 1997). If the
actin filaments are experimentally extracted from the sarcomere, this
part of titin becomes elastic (Linke et al., 1997; Trombitas and Granzier,
1997). The network of titin-binding partners within the Z-disk is still
expanding (Filomena et al., 2021; Rudolph et al., 2020), also reflecting
the participation of Z-disk titin in multiple signaling pathways. In
summary, N-terminal titin is an integral part of the Z-disk that has both
structural and signaling functions.
1.5. I-band titin architecture and composition
The cDNA sequencing (Labeit and Kolmerer, 1995) was also instru­
mental in showing that elastic titin undergoes extensive alternative
splicing of exons (Fig. 1E, F) resulting in different-length titin isoforms.
In skeletal muscle, these isoforms are coined “N2A” (Fig. 1F), whereas
the heart has “N2B” and “N2BA” (Freiburg et al., 2000), the latter of
which was initially called “N2A” as well (Labeit and Kolmerer, 1995).
Both N2BA and N2A (but not N2B) exist in many lengths variants
because of the I-band splicing diversity. Human N2A titins express 312
exons or less, out of the 364 exons present in TTN (Fig. 1F). It was
important news that I-band titin contains up to ~ 100 Ig-domains, no
FN3-domain, and one or two large unique sequences (Labeit and Kol­
merer, 1995). One of them is expressed only in cardiac titin, the N2-B-
unique sequence (N2-Bus; ~570 residues), which is part of the N2-B
element coded by TTN-exon 49 (Fig. 1E). The other, even larger
unique sequence (up to ~ 2,200 residues) is the PEVK-segment named so
after its high content of proline, glutamic acid, valine and lysine resi­
dues. The PEVK-segment is an intrinsically disordered region organized
as repeating motifs of mostly 28 amino acids, each typically coded by a
single exon. PEVK encompasses as many as 114 exons in humans (TTN-
exons 112–225), although some never seem to be expressed in muscle
(Fig. 1F). Only the PEVK-exons 220–225 are constitutively expressed, all
others are differentially spliced.
The Ig-domain regions of I-band titin were classified into constitu­
tively expressed “proximal” and “distal” segments, complemented by a
differentially spliced “middle” Ig-segment (Fig. 1E, F) (Freiburg et al.,
2000; Labeit and Kolmerer, 1995). An I-band Ig-domain is almost always
encoded by a single TTN exon. These Ig-domains are usually arranged in
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Journal of Biomechanics 152 (2023) 111553
tandem and are connected by short linkers providing some flexibility
and extensibility (Improta et al., 1996), like a “carpenter’s ruler” (von
Castelmur et al., 2008). The name-giving region of the N2A (and N2BA)
titin isoforms, termed N2-A element, is located in-between the differ­
entially spliced Ig-domains and the PEVK-segment and is composed of
four Ig-domains plus intervening sequences (Labeit and Kolmerer,
1995). As discussed later, the N2-A element is a mechanical signaling
hub (Fig. 2).
A few titin exons were overlooked in the early cDNA-sequencing
studies but were added later (Bang et al., 2001). The most prominent
of these “novel” exons is Novex-3 (TTN-exon 48), which contains a
termination signal generating a short, Z-disk-anchored, titin isoform of
~ 650 kDa also called Novex-3 (Fig. 1F). This isoform (expressed in
skeletal and cardiac muscles) does not reach the A-band and probably is
not elastic. Novex-3 is involved in protein–protein interactions but
otherwise, is little characterized (Kellermayer et al., 2017). Modern
next-generation sequencing approaches have since been useful to the
field, to validate the canonical sequence of titin and its modifications
(Genomes Project., 2010).
1.6. The Cronos isoform of titin
Yet another muscle titin species was discovered much later, the
Cronos isoform (Zou et al., 2015). Cronos is under the control of an
internal, alternative promoter located upstream of exon 241 in human
TTN (Fig. 1F). Interestingly, Cronos is nearly absent in slow skeletal
muscles but prominently expressed in fast muscles (Swist et al., 2020;
Zou et al., 2015). In adult human hearts, Cronos makes up ~ 12 % of all
titin proteins (Fomin et al., 2021), whereas embryonic cardiomyocytes
express much more Cronos (Zaunbrecher et al., 2019). Like A-band titin,
Cronos may be a molecular ruler for the thick filaments as it is able to
support de-novo sarcomerogenesis in embryonic cardiomyocytes even
in the absence of full-length titins (Zaunbrecher et al., 2019). However,
Cronos cannot maintain the sarcomere structure in adult mouse skeletal
muscles depleted of the Z-disk-anchored titin isoforms (Swist et al.,
2020). The function of Cronos is still incompletely understood.
1.7. Diversity in titin elasticity and titin-based force
Arguably the best-known property of titin is the elasticity and ability
to generate a “passive” force when stretched, as already proposed at the
time of its discovery (Maruyama, 1976). Evidence obtained during the
mid-1980 s to mid-1990 s confirmed that titin must be responsible for at
least part of the developed passive tension when nonactivated skeletal
muscle is stretched (Funatsu et al., 1990; Granzier and Wang, 1993;
Magid and Law, 1985; Wang et al., 1991, 1993) and for restoring the
sarcomeres to their initial length after a physiological stretch (Trombitas
et al., 1993; Wang et al., 1991). Even before the titin sequence was
published, different muscles were found to have a different titin protein
size, which is inversely correlated with the magnitude of passive tension
the muscle develops (Horowits, 1992; Wang et al., 1991). When I
worked as a postdoc in the laboratory of Prof. Gerald Pollack in the early
1990 s, I learned how to measure the forces generated by a single
myofibril (Fig. 3). It turned out that an isolated cardiac myofibril is
usually stiffer than an isolated skeletal myofibril (Bartoo et al., 1993;
Linke et al., 1994). We proposed that cardiac myofibrils — probably
their titin filaments — are responsible for most of the passive tension
even in multicellular cardiac specimens that contain extracellular matrix
proteins (Linke et al., 1994). Later we reported that single myofibrils of
different skeletal muscle types also differ in their passive sarcomere
length (SL)-tension relationships: sarcomeres of slow-type muscles (e.g.,
soleus) are typically less stiff than those of fast-type muscles (e.g.,
psoas), which in turn are less stiff than cardiac sarcomeres (Fig. 3) (Linke
et al., 1998a; Linke et al., 1996; Linke et al., 1999). The primary
sequence of titin (Labeit and Kolmerer, 1995) explained this elastic di­
versity, demonstrating that I-band titin is longer in soleus than in psoas
W.A. Linke
Fig. 3. Diversity in titin-based passive tension among muscle types. Pas­
sive sarcomere-length tension relationships recorded on passively stretched
single myofibrils from different striated muscle types (rat). Quasi steady-state
“elastic” tension is shown. Inset: Single myofibril suspended for mechanical
measurements; scale bar, 3 µm. Modified from Neagoe et al., 2003.
muscle, whereas cardiac N2B has the shortest I-band titin. The differ­
ences are brought about by splicing-in more or less middle Ig-domains
and PEVK-repeats. For details, I refer to relevant original articles and
reviews published at that time (Freiburg et al., 2000; Granzier et al.,
2000; Linke, 2000; Neagoe et al., 2003).
The great elastic diversity of titin and its relevance to sarcomere
stiffness has since been revealed in many studies. For instance, each
skeletal muscle may have its “personalized” titin-isoform length and
titin-based stiffness (Li et al., 2012; Prado et al., 2005; Tirrell et al.,
2012). However, titin isoform-based stiffness and extracellular-matrix
stiffness can be inversely correlated to one another (Prado et al.,
2005). Furthermore, developing hearts and muscles switch their I-band
titin springs from long/compliant in fetal tissues to shorter/stiffer during
and after birth (Lahmers et al., 2004; Opitz et al., 2004; Ottenheijm
et al., 2009; Warren et al., 2004). The cardiac titin-isoform composition
also varies in the different chambers of the heart (Cazorla et al., 2000;
Neagoe et al., 2003) and among different adult species, in that short/stiff
titins predominate in small rodent hearts and longer/softer titins in large
animals (Cazorla et al., 2000; Neagoe et al., 2003). Moreover, the N2BA:
N2B titin-isoform ratio is affected by hormones like insulin or thyroid
hormone (Krüger et al., 2010; Krüger et al., 2008) and can be altered in
heart disease (Makarenko et al., 2004; Nagueh et al., 2004; Neagoe
et al., 2002; Wu et al., 2002). A key factor regulating titin splicing is
RNA-binding motif 20 (RBM20; Guo et al. (2012)). This splicing factor
regulates > 50 different proteins in the heart, including titin, but it also
splices titin in skeletal muscles (Buck et al., 2014). Normal RBM20 ac­
tivity promotes the shorter/stiffer titins, such as N2B in the heart, but
mutations in RBM20 (or deletion) repress I-band splicing, a giant titin
(>3.8 MDa) remains (Guo et al., 2012), and heart disease can develop
(Koelemen et al., 2021). In conclusion, titin’s elastic diversity suggests
that titin-based stiffness is individually fine-tuned to allow the proper
mechanical function of the different striated muscle types in diverse
situations throughout the lifespan.
1.8. Exploring the molecular mechanisms of titin elasticity
In the early 1990 s, A-band titin was suggested to be recruited to the
elastic titin segment during higher physiological excursions, when the
titin-myosin bonds at the thick-filament tips become disrupted (“yield
point”; (Wang et al., 1991, 1993)). However, the yield point occurs at ~
3.8–4.5 µm SL, depending on muscle type, which is outside of the
physiological SL range (Linke et al., 1996). Other models of titin
extensibility assumed that the 7–8 stranded β-sheets of the Ig/FN3-
domains can unfold (Pfuhl and Pastore, 1995; Soteriou et al., 1993),
explaining titin elasticity (Erickson, 1994). In addition, the short linker
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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
single myofibrils (Fig. 4B) (Linke et al., 1998a). Importantly, the
sequential extension of Ig- and PEVK-segments occurs at physiological
forces and SLs (Fig. 4C). A twist is that some muscle fibers co-express
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
sequential titin-extension model (Gautel and Goulding, 1996; Linke
et al., 1996) was quickly confirmed (Linke et al., 1998a; Linke et al.,
1998b; Trombitas et al., 1998; Tskhovrebova and Trinick, 1997), it
remained unclear for some time whether the Ig-domains of I-band titin
unfold during physiological stretching.
Much of this discussion was based on the findings of three landmark
papers published back-to-back in 1997, which measured the force-
extension relationships of isolated titin molecules or recombinant titin
fragments using the atomic force microscope (AFM; Rief et al. (1997)) or
optical tweezers (Kellermayer et al., 1997; Tskhovrebova et al., 1997).
These studies found that titin Ig-domains unfold under relatively high
forces (150–250 pN in AFM force-extension experiments; Fig. 4D) and
concluded that these unfolding forces are probably too high to be
physiological. Interestingly, the unfolded titin domains refolded under
forces of a few piconewtons. The outstanding relevance of these papers
is based on their recognition of titin as a polymer whose elasticity can
largely be described as that of an entropic spring. Moreover, they firmly
established the new field of single-molecule protein mechanics. These
experiments were also complemented with mathematical modelling,
especially steered molecular dynamics simulations, providing atomic-
level detail of domain unfolding events (comprehensively reviewed by
Hsin et al. (2011)). Since this review is not the place to recapitulate the
various important implications of these studies, I refer to relevant re­
views (Fisher et al., 2000; Granzier et al., 2002; Kellermayer and Grama,
2002; Linke and Fernandez, 2002; Linke and Grützner, 2008). Collec­
tively, these and subsequent single-molecule experiments on titin (e.g.,
Li et al. (2002); Marszalek et al. (1999); Nagy et al. (2005); Oberhauser
et al. (2001); Watanabe et al. (2002)) validated the sequential titin-
extension hypothesis (Fig. 4C), established and extended the concept
of titin as a molecular spring whose elasticity is entropically driven but
also has enthalpic components — e.g., entropic elasticity cannot explain
viscoelastic behavior but Ig-domain unfolding can (Minajeva et al.,
2001) — and thus, answered crucial questions about the molecular
mechanisms of titin elasticity.
The issue of “physiological” titin domain unfolding was re-addressed
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
time (Fig. 4E). Under these “physiological” stretch conditions, repetitive
lengthening-shortening steps of ~ 10 nm were observed, clearly indi­
cating Ig-domain unfolding-refolding transitions (Fig. 4E) (Rivas-Pardo
et al., 2016). Similar events with step sizes of ~ 10–13 nm appeared in
immunolabeled single psoas myofibrils after a quick stretch to ~ 3.1 µm
SL, corresponding to ~ 6 pN/titin (Rivas-Pardo et al., 2016). Because
shortening against a force means work production, Ig-domain refolding
was proposed to be an “extra kick” in the power output of shortening
muscle (Martonfalvi et al., 2017; Rivas-Pardo et al., 2016). While such a
W.A. Linke Journal of Biomechanics 152 (2023) 111553

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

6
W.A. Linke
effect of the titin modification on the sarcomeres.
An elegant model to quantify titin stiffness is the titin-cleavage
mouse, in which a protease from the tobacco etch virus (TEV) can be
used to cleave I-band titin within a genetic cassette cloned into the distal
Ig-region (Rivas-Pardo et al., 2020). In homozygous mutant (Hom) mice,
100 % titin can be cleaved specifically and acutely by TEV, whereas titin
is not cleaved by TEV in wildtype (Wt) mice. We employed this model to
precisely measure the titin contribution to the SL-dependent passive
elastic force of permeabilized psoas muscle fiber bundles (Li et al.,
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
was mainly attributed to the collagen fibers (still present in these sam­
ples), whose relative contribution to stiffness increased above ~ 3.2 µm
SL. It will be interesting to also use this mouse model to clarify the
relevance of titin for the passive force of whole skeletal muscle in vivo,
which has recently been claimed to be overwhelmingly determined by
Fig. 5. Contribution of titin to passive tension and the stability of muscle
fibers. A) Passive sarcomere length (SL) - elastic force relationships of per­
meabilized psoas fiber bundles from the titin-cleavage mouse model. Wildtype
(Wt) fibers are not cleaved by tobacco etch virus protease (TEV) and force re­
mains unaltered (left). In homozygous mutant fibers (Hom), 100 % titin
cleavage by TEV causes a drop in force at all SLs (right). Red numbers indicate
statistically significant decrease in mean force at a given SL (%). B) Electron
micrographs of Wt (left) and Hom (right) muscle fibers following TEV treatment
and passive stretch experiments; scale bars, 1 µm. C) Interpretation of ultra­
structural changes in passively stretched sarcomeres with intact titin (top, Wt)
or after 100 % titin cleavage by TEV (bottom, Hom). Some elastic titin remains
bound to the thin filaments, most of it retracts to the Z-disk. Modified from Li
et al., 2020.
7
Journal of Biomechanics 152 (2023) 111553
the extracellular matrix (Lieber and Binder-Markey, 2021). In our hands,
an additional observation in 100 % titin-cleaved and stretched muscle
fibers was the misalignment of Z-disks and A-bands (Fig. 5B, C), sug­
gesting that titin stiffness — even if low, e.g., at 2.4 µm SL (Fig. 5B) — is
required to keep the sarcomeres organized and aligned during passive
stretching (Li et al., 2020). In summary, while the contribution of titin
stiffness to total passive muscle stiffness requires further characteriza­
tion, it is clear that titin is the main determinant of passive elastic (and
also viscous!) forces over the physiological SL range in fiber bundles.
1.10. Regulation of titin-based stiffness
A fascinating fact is that the stiffness of titin in muscle fibers is not
constant. As discussed, it depends on the titin-isoform pattern and I-
band spring composition. However, there are ways to also modulate titin
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),
especially small HSPs like αB-crystallin (CRYAB) and HSP27, to I-band
titin (N2-Bus, N2-A, and Ig-domains; (Bullard et al., 2004; Kötter et al.,
2014). In skeletal muscles under diverse stress conditions, including
disease or intense exercise, CRYAB, HSP27, and the ATP-dependent
chaperone HSP90, translocate from the cytosol to (unfolded) I-band
titin domains, probably to protect them from aggregation and quick
degradation (Fig. 2) (Kötter et al., 2014; Unger et al., 2017), while
slightly increasing titin-based passive stiffness (Bullard et al., 2004;
Unger et al., 2017). The binding of HSP90 to the N2-A element is
regulated by a co-chaperone, the methyltransferase Smyd2 (Donlin
et al., 2012). Another way to increase titin stiffness is through Ca2+-
binding to the differentially spliced PEVK sub-segment, which has a net
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
lengths, ensuring optimal actin-myosin interactions. Recently, my group
has used the titin-cleavage mouse aiming to prove this property of titin,
while avoiding the unspecific effects of radiation damage (Li et al.,
2020). We used TEV to specifically cleave 100 % titin in permeabilized,
stretched muscle fibers and then Ca2+-activated the muscle maximally
(Fig. 6). We found that thick filament bundles or individual thick fila­
ments moved uncontrolledly toward the Z-disks during contraction,
which reduced active force. The damage to the sarcomeres progressed
with the duration of contraction, causing thick filaments to translocate
through the Z-disk, misalign, and even shed myosin molecules from their
tips, thus verifying and extending the findings of Horowits and Podolsky
(1988). Damage tended to be higher when sarcomeres contracted at
longer SLs. Another conclusion of our work was that the spring stiffness
of intact titin in resting muscle is not sufficient to perform the A-band
centering function: it must increase considerably (~4-fold) during
activation (Li et al., 2020). Such an increase has indeed been predicted
(Nishikawa, 2020) or measured before (Leonard and Herzog, 2010) and
additional evidence in support of elevated “non-crossbridge stiffness”
has been provided in active muscle mechanics experiments (Herzog,
2018; Powers et al., 2020; Rassier et al., 2015). The reasons behind the
titin stiffness increase in stretched and activated muscle have not yet
been identified, despite interesting candidate mechanisms, such as
actin-titin interactions (Freundt and Linke, 2019; Herzog, 2018; Hessel
and Linke, 2021; Nishikawa, 2020; Tomalka et al., 2020).
A related issue is the origin of residual force enhancement (RFE), a
phenomenon typically associated with eccentric muscle activity and
characterized by increased steady-state force following active muscle
stretching (Herzog, 2018). An impressive amount of work from the
Herzog, Nishikawa, and other labs has suggested a titin-stiffening effect
as the most probable explanation of RFE (e.g., Boldt et al., 2020;
Fukutani and Herzog, 2020; Herzog and Leonard, 2002; Leonard and
Herzog, 2010; Nishikawa, 2020; Nishikawa et al., 2020a; Powers et al.,
Fig. 6. Deficits in sarcomere ultrastructure arising during active contra­
tion following specific cleavage of the titin springs. Homozygous mutant
sarcomeres (length, ~2.7 µm) from the titin-cleavage mouse before treatment
with tobacco etch virus protease (Hom-TEV) and following 100 % titin cleavage
by TEV (Hom + TEV) in the passive sarcomere or when maximally activated in
Ca2+-containing buffer. Modified from Li et al., 2020.
2014; Rassier et al., 2005; Schappacher-Tilp et al., 2015). The molecular
basis of the proposed acute titin stiffening is subject to an ongoing
debate but actin-titin interactions involving the PEVK-N2-A region and
possible effects of Ca2+ on these parameters are discussed.
Finally, titin also plays a critical role in length-dependent activation
(LDA), which is characterized by increased contractile force after a
stretch of muscle (or heart; the molecular basis of the Frank-Starling
law). Initial evidence for titin as a factor in LDA of cardiac muscle was
provided >20 years ago (Cazorla et al., 2001; Fukuda et al., 2001) and
has since been extensively confirmed. However, LDA also occurs in
skeletal muscles and is, at least in part, titin-dependent (Hessel et al.,
2022; Irving et al., 2011; Mateja et al., 2013). We have recently gained
insight into the underlying mechanisms of titin-dependent LDA by
comparing thick and thin filament protein periodicities in small-angle X-
ray diffraction measurements on permeabilized muscle fibers that were
passively stretched before and after 50 % targeted titin cleavage (using
the heterozygous titin-cleavage mouse; Hessel et al. (2022)). The acute
reduction in titin stiffness blunted the length-dependent priming of both
thin and thick filaments, as judged from characteristic changes in X-ray
reflection patterns. Moreover, active force measurements demonstrated
reduced myofilament Ca2+ sensitivity after 50 % titin cleavage (Hessel
et al., 2022). We concluded that the stretched titin springs pulling on the
myosin filaments convert the myosin heads from “off” to “on”, priming
them for interaction with actin (Fig. 7). The concomitant priming of
actin and troponin requires the presence of non-crossbridge links

8
W.A. Linke
between the thick and thin filaments in the passive state, possibly
MyBPC. These findings strongly support a role for titin in LDA.
In summary, titin has evolved from a “ghost-like” filament to a mere
passive spring to an essential protein that determines the organization of
the sarcomere, adjusts its stiffness according to the functional re­
quirements of striated muscles, and tunes active contraction. Clearly, the
nearly 50 year-long titin story has not yet come to an end.
CRediT authorship contribution statement
Wolfgang A. Linke: Conceptualization, Funding acquisition, Inves­
tigation, Project administration, Resources, Supervision, Writing –
original draft, Writing – review & editing.
Declaration of Competing Interest
The author declares that he has no known competing financial in­
terests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
I thank the past and present members of my laboratory involved in
the studies described. My research is supported by the German Research
Foundation (SFB1002A08), IZKF Münster (Li1/029/20), the German
Center for Cardiovascular Research, and the European Union.
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Fig. 7. Length-dependent activation triggered by
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