International review of cell and molecular biology, volume 321

VOLUME THREE HUNDRED AND TWENTY ONE

INTERNATIONAL REVIEW OF
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International Review of Cell
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JAMES F. DANIELLI
KWANG W. JEON
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JONATHAN JARVIK

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VOLUME THREE HUNDRED AND TWENTY ONE

INTERNATIONAL REVIEW OF
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ISBN: 978-0-12-804707-1
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CONTRIBUTORS
Sara Aspengren
Department of Biology and Environmental Sciences, University of Gothenburg, Go¨teborg,
Sweden
Lidia Bakota
Department of Neurobiology, University of Osnabru¨ck, Osnabru¨ck, Germany
Roland Brandt
Department of Neurobiology, University of Osnabru¨ck, Osnabru¨ck, Germany
Elizabeth Calzada
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore,
MD, USA
Karen L. Cheney
School of Biological Sciences, University of Queensland, Brisbane, Australia
Steven M. Claypool
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore,
MD, USA
Yusuke Ito
Plant Molecular Breeding Laboratory, Bioscience and Biotechnology Center, Nagoya
University, Nagoya, Japan
Jian-Ping Jin
Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA
Henriikka Kentala
Minerva Foundation Institute for Medical Research, Biomedicum 2U, Helsinki, Finland
Makoto Matsuoka
Plant Molecular Breeding Laboratory, Bioscience and Biotechnology Center, Nagoya
University, Nagoya, Japan
Younes Medkour
Department of Biology, Concordia University, Montreal, Quebec, Canada

Yoichi Morinaka
Plant Molecular Breeding Laboratory, Bioscience and Biotechnology Center, Nagoya
University, Nagoya, Japan
Vesa M. Olkkonen
Minerva Foundation Institute for Medical Research, Biomedicum 2U, Helsinki, Finland

ix


x

Contributors

Ouma Onguka
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore,
MD, USA
Reynante Ordonio
Plant Molecular Breeding Laboratory, Bioscience and Biotechnology Center, Nagoya
University, Nagoya, Japan
Lore`ne Penazzi
Department of Neurobiology, University of Osnabru¨ck, Osnabru¨ck, Germany
Takashi Sazuka
Plant Molecular Breeding Laboratory, Bioscience and Biotechnology Center, Nagoya
University, Nagoya, Japan
Helen Nilsson Sko¨ld
Sven Loven Centre for Marine Sciences—Kristineberg, University of Gothenburg,
Fiskeba¨ckskil, Sweden
Veronika Svistkova
Department of Biology, Concordia University, Montreal, Quebec, Canada
Vladimir I. Titorenko

Department of Biology, Concordia University, Montreal, Quebec, Canada
Margareta Wallin
Department of Biology and Environmental Sciences, University of Gothenburg, Go¨teborg,
Sweden
Marion Weber-Boyvat
Minerva Foundation Institute for Medical Research, Biomedicum 2U, Helsinki, Finland


CHAPTER ONE

Evolution, Regulation,
and Function of N-terminal
Variable Region of Troponin T:
Modulation of Muscle Contractility
and Beyond
Jian-Ping Jin*
Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA

*E-mail:

Contents
Introduction
Molecular Structure of Troponin T
Evolution of Troponin T Isoform Genes
Alternative Splicing
Developmental Regulations
Posttranslational Modifications
6.1 Phosphorylation
6.2 Restrictive Proteolysis
7. Conclusion and Perspectives

Acknowledgments
References

2
3
6
10
15
19
19
20
22
22
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1.
2.
3.
4.
5.
6.

Abstract
Troponin T (TnT) is the tropomyosin-binding and thin filament-anchoring subunit of
the troponin complex in skeletal and cardiac muscles. At the center of the sarcomeric
thin filament regulatory system of striated muscles, TnT plays an essential role in
transducing Ca2+ signals in the regulation of contraction. Having emerged predating
the history of vertebrates, TnT has gone through more than 500 million years of
evolution that resulted in three muscle-type-specific isoforms and numerous alternative RNA splicing variants. The N-terminal region of TnT is a hypervariable structure
responsible for the differences among the TnT isoforms and splice forms. This focused

review summarizes our current knowledge of the molecular evolution of the Nterminal variable region and its role in the structure and function of TnT. In addition

International Review of Cell and Molecular Biology, Volume 321
ISSN 1937-6448
/>
© 2016 Elsevier Inc.
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2

Jian-Ping Jin

to the physiologic and pathophysiologic significances in modifying the contractility of
skeletal and cardiac muscles during development and in adaptation to stress and
disease conditions, the hyperplasticity of the N-terminal region of TnT demonstrates
an informative example for the evolution of protein three-dimensional structure and
provides insights into the molecular evolution and functional potential of proteins.

1. INTRODUCTION
The contractile machinery of striated muscles (represented by skeletal
and cardiac muscles of vertebrates) is the myofibrils that consist of tandem
repeats of sarcomeres. A sarcomere is composed of overlapping myosin thick
filaments and actin thin filaments. Contraction is powered by actin-activated
myosin ATPase-catalyzed ATP hydrolysis during actomyosin cross-bridge
cycling. This process is regulated by the thin filament-associated regulatory
proteins troponin under the control of cytosolic Ca2+ (Gordon et al., 2000).
Residing at ∼37-nm intervals along the thin filament in the form of

F-actin-tropomyosin double helices (Galinska-Rakoczy et al., 2008;
Lehman et al., 2009; Ohtsuki et al., 1967), the troponin complex consists
of three protein subunits: the Ca2+-binding subunit troponin C (TnC1),
actomyosin ATPase-inhibiting subunit troponin I (TnI), and tropomyosinbinding subunit troponin T (TnT) (Greaser and Gergely, 1971). To convert
the cellular signal of cytosolic Ca2+ transient originated from sarcolemmal
electrical activity to myofilament movements during each excitation–
contraction–relaxation cycle, troponin functions through cooperative interactions among the three subunits and with tropomyosin (Gordon et al., 2000;
Tobacman, 1996). Whereas TnC is a relative of the calmodulin gene family
(Collins, 1991) and functions as the Ca2+ receptor of the thin filament regulatory system in striated muscle, TnI and TnT are striated-muscle-specific
proteins encoded by closely linked genes and have coevolved into three pairs
of fiber-type-specific isoforms (Chong and Jin, 2009; Jin et al., 2008).
In addition to anchoring the troponin complex to the thin filament, TnT
directly interacts with multiple proteins in the thin filament regulatory
system to play an organizer role in the troponin complex (Perry, 1998).
Through isoform gene regulation, alternative RNA splicing, and posttranslational modifications, structural and functional variations of TnT provide a
mechanism to modulate striated muscle contraction and relaxation.
To understand the structure–function relationship of TnT, this review
outlines the evolution of muscle type-specific TnT isoform genes, the


Modulation of Muscle Contractility and Beyond

3

multiple alternative splice forms, the developmental regulation of isoform
expression and alternative splicing, and the posttranslational modifications
during physiologic and pathophysiologic adaptations, with a focus on the Nterminal segment that is an evolutionarily diverged regulatory structure
(Chong and Jin, 2009; Jin et al., 2008; Wei and Jin, 2011). For background
information, comprehensive summaries of striated muscle thin filament
regulation and the functions of TnC, TnI, and tropomyosin can be found

in several previously published reviews (Collins, 1991; Gordon et al., 2000;
Jin et al., 2008; Perry, 1998, 1999, 2001; Solaro and Rarick, 1998;
Tobacman, 1996; Wei and Jin, 2011; Sheng and Jin, 2014).

2. MOLECULAR STRUCTURE OF TROPONIN T
TnT is a 30–35-kDa protein. The sizes of vertebrate TnT with
sequence information available range from 223 to 305 amino acids. This
large size variation is almost entirely due to the variable length of the Nterminal region, from nearly absent in certain fish fast skeletal muscle TnT to
more than 70 amino acids long in avian and mammalian cardiac TnT (Jin
et al., 2008; Wei and Jin, 2011). The hypervariable nature of the N-terminal
domain of TnT is further demonstrated by the presence of 4–9 repeating
sequence motifs in the breast muscle fast TnTof avian orders Galliformes and
Craciformes (Jin and Smillie, 1994). These five amino acid repeats form a
cluster of high-affinity transition metal binding sites that are only found in
the adult breast muscle of these birds (Jin and Samanez, 2001; Ogut et al.,
1999). While the N-terminal region of TnT is hypervariable in length and
amino acid sequences, the amino acid sequences of the middle and Cterminal regions of TnT are highly conserved among the three muscletype-specific isoforms and across vertebrate species (Jin et al., 2008; Wei
and Jin, 2011).
Electron microscopic studies showed that the TnT molecule has an
extended conformation (Cabral-Lilly et al., 1997; Wendt et al., 1997).
The functional domains of TnT have been extensively studied using protein fragments generated from limited chymotryptic and CNBr digestions.
Protein-binding studies found that the ∼100 amino acids C-terminal chymotryptic fragment T2 interacts with TnI and TnC and binds to the
middle region of tropomyosin (Heeley et al., 1987; Schaertl et al., 1995).
The chymotryptic fragment T1 that contains both the N-terminal variable
region and the middle conserved region of TnT binds the head–tail


4

Jian-Ping Jin

junction of tropomyosins in the actin thin filament (Heeley et al., 1987).
The tropomyosin-binding activity of the T1 fragment resides in the 81
amino acids CNBr fragment CB2 of rabbit fast skeletal muscle TnT, which
represents the middle conserved region of TnT. The N-terminal segment
of TnT (e.g., the CNBr fragment CB3 in rabbit fast skeletal muscle TnT) is
the hypervariable region and does not bind any known thin filament
proteins in the sarcomere (Perry, 1998).
Consistent with the protein-binding data, X-ray crystallography determined the partial structure of cardiac and skeletal muscle troponin complex
showing that the associations of TnTwith TnI and TnC are through the Cterminal T2 region (Takeda et al., 2003; Vinogradova et al., 2005). However,
the crystallography data only determined the structure for a portion of the
TnT–T2 region in the troponin complex. The entire T1 region and the very
C-terminal 13 amino acids of TnT were missing from the resolved highresolution structures (Takeda et al., 2003; Vinogradova et al., 2005). The 13
amino acid C-terminal end segment encoded by the last exon of the TnT
gene is highly conserved among isoforms and across species (Jin et al., 2008;
Wei and Jin, 2011). Deletion of the C-terminal 57 amino acids of fast TnT
(Jha et al., 1996) or slow TnT (Jin and Chong, 2010) had no significant effect
on the binding affinity of TnT for tropomyosin. However, point mutations
in this segment have been found to cause familial hypertrophic cardiomyopathy (Sheng and Jin, 2014); thus, its role in the structure and function of
TnT remains to be investigated.
The high-resolution structural data showed that the main TnT–TnI
interface in the troponin complex is a coiled-coil structure (i.e., the I-T
arm) formed by the segments of L224–V274 in cardiac TnT and F90–R136 in
cardiac TnI in human cardiac troponin complex (Takeda et al., 2003) or
E199–Q245 in fast TnTand G55–L102 in fast TnI in chicken fast skeletal muscle
troponin (Vinogradova et al., 2005). The amino acid sequences of TnT and
TnI in this coiled-coil interface are both highly conserved among isoforms
and across vertebrate species (Jin et al., 2008; Wei and Jin, 2011).
Whereas gross mapping of the tropomyosin-binding sites of TnT using
the chymotrypsin and CNBr fragments had served in guiding the studies of

TnT function and thin filament regulation of muscle contraction for over
three decades (Perry, 1998), the precise localizations of the two tropomyosinbinding sites of TnT were not determined until recently using genetically
engineered TnT fragments (Jin and Chong, 2010). Analysis of serial deletions of TnT protein and mapping using site-specific monoclonal antibody
epitope probes showed that the T1 region tropomyosin-binding site of TnT


Modulation of Muscle Contractility and Beyond

5

involving a large content of α-helix interactions (Pearlstone et al., 1976,
1977) corresponds mainly to a 39 amino acids segment in the beginning of
the conserved middle region (Jin and Chong, 2010). On the other hand, the
T2 region tropomyosin-binding site depends on a segment of 25 amino acids
near the very beginning of the T2 fragment (Jin and Chong, 2010). Amino
acid sequences in the two tropomyosin-binding sites are both highly conserved in the three muscle-type TnT isoforms and across vertebrate species
(Jin et al., 2008; Wei and Jin, 2011).
Although the N-terminal variable region of TnT does not contain
binding sites for TnI, TnC, or tropomyosin (Ohtsuki et al., 1984; Pan
et al., 1991; Pearlstone and Smillie, 1982), its structure is regulated by
alternative splicing during late embryonic and early postnatal development
of the heart (Jin and Lin, 1988) and skeletal muscles (Wang and Jin, 1997),
and in pathologic adaptation (Larsson et al., 2008). These developmental and
adaptive regulations suggested functional significances of the N-terminal
variable region of TnT. To investigate the molecular mechanism for the
N-terminal variable region to affect TnT function, we developed an epitope
conformational analysis using monoclonal antibodies recognizing the middle
and C-terminal regions of TnT as three-dimensional structure-sensitive
probes. The studies demonstrated that local structural changes in the Nterminal region of TnT, such as that induced by Zn2+-binding to a transition
metal ion (Cu(II), Ni(II), Co(II), and Zn(II)) binding cluster in an α-helix in

the N-terminal variable region of chicken breast muscle fast TnT, and the
alternative splicing of N-terminal coding exons in cardiac TnT, altered the
structural conformation of remote regions and altered the binding affinity for
TnI and tropomyosin (Biesiadecki et al., 2007; Ogut and Jin, 1996; Wang
and Jin, 1998). Fluorescence spectrometry studies further demonstrated that
Cu2+-binding to the N-terminal metal-binding cluster in chicken breast
muscle fast TnT altered the fluorescence intensity and anisotropy of Trp234,
Trp236, Trp285, and fluorescein-labeled Cys263 in the C-terminal region
(Jin and Root, 2000). These long-range conformational effects indicate that
the N-terminal variable region of TnT plays a role in modulating the overall
molecular conformation and function of TnT.
The structural and functional domains of TnTare summarized in Figure 1.
In addition to the hypervariable N-terminal region, there are two other
variable regions in the TnT polypeptide chain. The C-terminal region of
fast skeletal muscle TnT contains a segment of 13 amino acids encoded by a
pair of mutually exclusive exons (exons 16 and 17). This region also shows
diversity between mammalian and avian cardiac TnT, where the avian cardiac


6

Jian-Ping Jin

TnC
HO

OC

Ch
ym

otr

Restricted
calpain I cleavage

yps

in c

lea
vag

Tnl
e

TnT
H2N N-terminal variable region
Tropomyosin-binding site 1

T1 T2

Tropomyosin-binding site 2

Figure 1 Structural and functional domains of TnT. The diagram summarizes the
structural and functional regions of TnT. The high-resolution structure of partial
troponin complex including a C-terminal segment of TnT that interacts with TnI and
TnC is redrawn from published crystallography data (Takeda et al., 2003). The arrows
indicate the chymotryptic cleavage site between the T1 and T2 fragments (Perry, 1998)
and the calpain I cleavage site for the selective removal of the N-terminal variable region

of cardiac TnT (Zhang et al., 2006). The two tropomyosin-binding segments (Jin and
Chong, 2010) are also outlined.

TnT gene contains an additional exon encoding two amino acids (Cooper and
Ordahl, 1985). The C-terminal variable region of TnT resides in the
TnI–TnT interface in troponin complex and is in the proximity of TnC
(Takeda et al., 2003; Vinogradova et al., 2005), whereas its functional significance and the regulation of its alternative splicing require more investigation.
There is another minor variable region between the middle and Cterminal regions of TnT (i.e., between the T1 and T2 fragments), where
an alternatively spliced exon (exon 13) is found in mammalian cardiac TnT
encoding a short segment of 2 or 3 amino acids (Jin et al., 1992, 1996). The
alternative splicing of this exon involves exclusion, complete, and partial
inclusions (Jin et al., 1996). The functional significance of this minor variable
region and the regulation of its alternative splicing also remain to be
investigated.

3. EVOLUTION OF TROPONIN T ISOFORM GENES
Three homologous genes have evolved in mammalian and avian species
encoding TnT isoforms in cardiac muscle (TNNT2), slow skeletal muscle


Modulation of Muscle Contractility and Beyond

7

(TNNT1), and fast skeletal muscle (TNNT3) (Breitbart and Nadal-Ginard,
1986; Cooper and Ordahl, 1985; Farza et al., 1998; Hirao et al., 2004; Huang
et al., 1999b; Jin et al., 1992). Expression of the three TnT isoform genes in
adult cardiac and skeletal muscles is controlled rather strictly in a muscle fiber
type-specific manner. Knockout of the cardiac TnT gene resulted in embryonic lethality (Nishii et al., 2008). A nonsense mutation in human slow TnT
gene that truncates the protein at amino acid 180 and deletes the TnI- and

TnC-binding sites together with one of the tropomyosin-binding sites in the
C-terminal T2 region (Jin et al., 2003; Johnston et al., 2000) resulting in the
loss of myofilament incorporation and rapid degradation of slow TnT1–179 in
the muscle cells (Jin et al., 2003; Wang et al., 2005) and a clinical phenotype of
recessive nemaline myopathy with infantile lethality (Johnston et al., 2000).
Therefore, the three TnT isoforms play nonredundantly critical roles in the
three types of striated muscle.
The primary structural diversity of the three muscle fiber type-specific
TnT isoforms is mainly in the N-terminal region (Chong and Jin, 2009; Jin
et al., 2008; Wei and Jin, 2011; Sheng and Jin, 2014). This observation is
consistent with the regulatory function of the N-terminal variable region
that provides a structural basis for adaptation to various functional demands
in different types of muscle, in different species, at different stages of development, and under pathologic conditions. On the other hand, the middle
and C-terminal regions of TnTare highly conserved among the three muscle
type-specific TnT isoforms and across vertebrate species.
Investigating the evolutionary lineage of the three TnT isoform genes
helps to understand the structure–function relationship of TnT as well as the
physiologic significance of the N-terminal hypervariable region. Material
remains of ancestor nucleotides and proteins are largely unavailable for
evolutionary studies, thus, like other molecular evolutionary biology studies,
nucleotide and amino acid sequence comparisons among homologous TnT
isoform genes in present-day organisms were employed to provide the core
of our current knowledge for the molecular evolution of TnT. It is worth
emphasizing that the variation in protein three-dimensional structure is a
basis for functional diversity. Therefore, the study of the evolution of threedimensional structures for TnT isoforms is a novel approach to enrich our
knowledge on troponin function and the thin filament regulation of muscle
contraction.
Using monoclonal antibodies as site-specific epitope probes, we detected
the restoration of ancestor-like conformation in TnT after removing certain
evolutionarily added “suppressor” structure, for example, the N-terminal

8

Jian-Ping Jin

variable segment. The findings demonstrate that TnT protein has the potential
of restoring ancestral conformations that have been allosterically suppressed by
the evolutionary addition of a modulatory structure. The results revealed
three-dimensional structural evidence for the evolutionary relationship
between TnI and TnT, two subunits of the troponin complex, and among
the three muscle fiber type-specific TnT isoforms (Chong and Jin, 2009).
Consistent with sequence analysis that suggested a distant homology
of the genes encoding TnI and TnT, the epitope analyses demonstrated
restoration of TnI-like three-dimensional structures in TnT, supporting that
these two subunits of troponin arose from a TnI-like ancestor protein
(Chong and Jin, 2009). This common ancestor would have had functions
in both anchoring to the actin-tropomyosin filament and inhibiting myosin
ATPase. TnI and TnT have diverged prior to the emerging of vertebrates
(Chong and Jin, 2009). It remains to be investigated whether any present-day
protein could represent the common ancestor of TnI and TnT, possibly in
invertebrate species.
Further supporting the notion that TnI and TnT genes are duplicates of a
common ancestral gene, TnI is also present in three muscle fiber type isoforms and the six TnI and TnT isoform genes are closely linked in three pairs
(fast TnI–fast TnT, slow TnI–cardiac TnT, and cardiac TnI–slow TnT) in the
genome of vertebrates (Chong and Jin, 2009; Jin et al., 2008). Embryonic
cardiac muscle expresses solely slow skeletal muscle TnI that is replaced by
cardiac TnI during late embryonic and early postnatal development (Jin,
1996; Saggin et al., 1989). The functional pairing of slow TnI and cardiac
TnT in embryonic heart indicates that the evolutionarily linked TnI–TnT

gene pairs, including the seemingly scrambled slow TnI–cardiac TnT and
cardiac TnI–slow TnT gene pairs, represent originally functional linkages.
In addition to the genomic linkages, TnI and TnT also have structural
alikeness that supports their origination by gene duplication. Like the structure of TnT, the N-terminal region of TnI is also a variable structure as
cardiac TnI has an evolutionarily additive N-terminal extension that is a
heart-specific regulator (Parmacek and Solaro, 2004; Perry, 1999) to fine
tune the conformation and function of cardiac TnI in physiologic and
pathophysiologic adaptations (Akhter et al., 2012; Jin et al., 2008).
By revealing suppressed three-dimensional structures, we further demonstrated an evolutionary lineage of fast to cardiac to slow TnT isoform
genes (Chong and Jin, 2009). Different from TnI and TnT that have evolved
into three isoforms for the three fiber types of vertebrate striated muscle,
TnC is present in only two isoforms: fast TnC (Gahlmann and Kedes, 1990)


9

Modulation of Muscle Contractility and Beyond

fTnT-like

cTnT-like

fTnT

cTnT

sTnT

Figure 2 Evolutionary lineage of TnT isoform genes. The evolutionary lineage of TnT
isoform genes is illustrated from the data of sequence analysis, immunological distance,

and experimental detection of evolutionarily suppressed conformational states (Chong
and Jin, 2009). Data suggested that TnT first emerged as an ancestral fast TnT gene.
A duplication event later resulted in the emergence of a cardiac TnT-like gene that was
further duplicated to give rise to the present-day cardiac TnT and slow TnT genes.

and slow–cardiac TnC (Parmacek and Leiden, 1989). The undifferentiated
utilization of the same TnC isoform in cardiac and slow skeletal muscles
supports the hypothesis that the emergence of the cardiac and slow TnI–TnT
gene pairs was a relatively recent event and the linked cardiac TnI–slow TnT
genes are the newest pair (Figure 2) (Chong and Jin, 2009). The latest
emergence of the cardiac TnI–slow TnT gene pair is supported by the
presence of the unique N-terminal extension in cardiac TnI (Chong and
Jin, 2009; Parmacek and Solaro, 2004). Summarized in Figure 2, this pattern
is consistent with the [fast skeletal (slow skeletal, cardiac)] phylogenetic
relationship indicated by sequence analysis of other muscle proteins (Oota
et al., 1999).
This novel experimental approach and the data identified structural evolutions that were critical to the emerging of diverged TnT isoforms, helping
to understand the origin as well as the functional potential of TnT structural
diversities. Sequence comparison demonstrated that each of the muscle typespecific TnT isoforms is more conserved across vertebrate species than that
among the three muscle-type TnT isoforms in the same species (Jin et al.,
1998, 2008). Such conservation pattern indicates that the evolution of TnT
isoform genes was driven primarily by early adaptations to the differentiated
functions of cardiac, fast, and slow skeletal muscles. The critical role of muscle
fiber type-specific TnT isoforms, the function of skeletal muscle, for example, slow TnT that is the newest isoform of TnT, is demonstrated by the


10

Jian-Ping Jin

human slow TnT Glu180 nonsense mutation (Jin et al., 2003) that causes
severe nemaline myopathy with infantile death (Johnston et al., 2000) and
confirmed by knocking down of the expression of slow TnT gene expression
in diaphragm muscle to produce atrophy, slow-to-fat fiber type switch, and
reduced resistance to fatigue in mouse muscles (Feng et al., 2009b).
We reported that the heart of adult toads Bufo expresses exclusively slow
skeletal muscle TnT instead of cardiac TnT while all other myofilament
proteins remain to be the cardiac isoforms including normal cardiac TnI
and cardiac myosin (Feng et al., 2012). This unique biochemical content of
toad cardiac muscle is correlated to a striking physiologic feature of toad
heart, that is, it is highly tolerant to large changes in the volume of body fluid
and blood between rainy and dry seasons (Boral and Deb, 1970) and much
more resistant to the loss of blood volume than that of the closely related frog
heart under experimental conditions (Deb et al., 1974). The aortic blood
flow rate of toad did not drop until a blood loss of more than 5% of the body
weight, whereas blood loss of 2% of the body weight caused a decline of
aortic blood flow rate in frog (Hillman and Withers, 1988). We demonstrated
that toad hearts had faster contractile and relaxation velocities and a significantly higher tolerance to afterload (Feng et al., 2012). These findings
indicate that the unique utilization of slow skeletal muscle TnT to replace
cardiac TnT in toad cardiac muscle was an evolutionary adaption with a
significant fitness value during natural selection, further supporting the
differentiated functionalities of TnT isoforms.
As discussed earlier, the main differences among the three muscle-type
TnT isoforms is in the N-terminal variable region (Jin et al., 2008; Wei and
Jin, 2011; Sheng and Jin, 2014) that fine tunes the molecular conformation
and function of TnT, thus represents a major driving force of the evolutionary diversity of TnT isoforms.

4. ALTERNATIVE SPLICING
Alternative RNA splicing generates multiple protein splice forms from
the transcripts of each of the three muscle type-specific TnT genes (Jin et al.,

2008; Wei and Jin, 2011). The mammalian cardiac TnT gene contains 14
constitutively expressed exons and 3 alternatively spliced exons, two of which
encode segments in the N-terminal variable region (Farza et al., 1998; Jin
et al., 1992, 1996). Exon 5 of cardiac TnT gene, which encodes 9 or 10
amino acids in the N-terminal variable region, is included in embryonic but


Modulation of Muscle Contractility and Beyond

11

not adult cardiac TnT (Jin and Lin, 1989). Exon 4 of cardiac TnT gene is
alternatively spliced independent of developmental stages (Jin et al., 1996).
The avian cardiac TnT gene contains 16 constitutively spliced exons and only
1 alternative exon (the embryonic exon 5) (Cooper and Ordahl, 1985).
Correspondingly, four mammalian and two avian cardiac TnT N-terminal
alternative splicing variants have been found in normal cardiac muscle.
Mammalian fast skeletal muscle TnT gene contains 19 exons, of which
exons 4, 5, 6, 7, 8, and a fetal exon encoding segments in the N-terminal
variable region are alternatively spliced (Breitbart and Nadal-Ginard, 1986;
Briggs and Schachat, 1993; Wang and Jin, 1997). These alternative exons are
not included or excluded randomly and not all possible splicing combinations are at a significant level detectable by cDNA cloning. Accordingly, only
13 mouse fast TnT mRNA variants and 11 chicken fast TnT mRNA variants
differing in the N-terminal variable region have been actually found with
sequence information to represent the splicing pathways for significant levels
of protein products and physiologic functions (Ogut and Jin, 1998; Smillie et
al., 1988; Wang and Jin, 1997).
In addition to exons 4–8, several unique N-terminal alternative coding
exons are found in avian fast skeletal muscle TnT genes. Seven P exons
located between exons 5 and 6 encode a unique Tx segment (Jin and

Samanez, 2001; Miyazaki et al., 1999; Smillie et al., 1988) consisting of
seven tandem repeats of pentapeptides (AHH[A/E]E) are found in chicken
fast TnT gene. A w exon and a y exon are found between exons 4–5 and 7–8,
respectively, further increasing the diversity of avian fast TnT (Schachat et al.,
1995). As discussed earlier, the Tx segment encoded by the P exons in the fast
TnT gene of birds in avian orders of Galliformes and Craciformes contains a
cluster of high-affinity transition metal ion binding sites (Jin and Smillie,
1994). No homologous counterpart was found in mammalian TnT genes
and the biologic significance of the Tx element remains to be investigated.
One of its specific physiologic functions is to serve as a Ca2+ reservoir (Zhang
et al., 2004), which may confer certain functions required for the avian flight
muscles.
The slow skeletal muscle TnT gene has a simpler structure than that of the
fast skeletal muscle and cardiac TnT genes. There are only 14 exons in the
slow TnT gene and one of which is alternatively spliced. With an exon–intron organization same as that of the mammalian slow TnT genes (∼9 kb),
chicken slow TnT gene is significantly smaller (3 kb) by having shorter
intron sequences (Hirao et al., 2004; Huang et al., 1999b). Alternative
splicing of exon 5 in the N-terminal region generates 2 variants of slow


12

Jian-Ping Jin

TnT (Gahlmann et al., 1987; Huang et al., 1999b; Jin et al., 1998). Splicing at
two alternative acceptor sites in intron 5 of mouse slow TnT gene further
generates a single amino acid variation in the exon 6-encoded segment
(Huang et al., 1999b). The same pattern was found for the intron 4–exon
5 splicing of chicken slow TnT gene transcript (Hirao et al., 2004).
The molecular mechanism that regulates the alternative splicing of

TnT mRNA is not fully understood. Both cis and trans regulatory factors
have been implicated to affect the alternative splicing of cardiac TnT (Ladd
and Cooper, 2002). Alternative splicing of fast TnT was found during
myogenesis. Muscle-specific trans regulatory factors were required for
appropriate splicing and incorporation of constitutive and alternative
exons of fast TnT during myotube differentiation in culture (Breitbart
and Nadal-Ginard, 1987).
The N-terminal alternatively spliced TnT variants have been shown
with functional impacts. Skinned fibers of adult chicken pectoral muscle
containing alternatively spliced fast TnT with more negatively charged
residues in the N-terminal variable region exhibited higher myofilament
calcium sensitivity than control muscle fibers containing alternatively
spliced TnT with less N-terminal negative charges (Ogut et al., 1999;
Reiser et al., 1992, 1996). When reconstituted into skinned cardiac muscle
strips, embryonic cardiac TnT with more negative N-terminal charges also
increased Ca2+ sensitivity of myosin ATPase and force development in
comparison to that of the less negatively charged adult cardiac TnT
(Gomes et al., 2002). Similarly, studies using reconstituted myofilaments
showed that the embryonic cardiac TnT produced higher Ca2+ sensitivity as
compared with that of adult cardiac TnT (Gomes et al., 2004). Embryonic
and neonatal cardiac muscle containing embryonic cardiac TnT exhibited
higher tolerance to acidosis (Solaro et al., 1988). In contrast, overexpression
of fast skeletal muscle TnT that has a less negatively charged N-terminal
segment than that of cardiac TnT decreased the tolerance to acidosis in
transgenic mouse cardiac muscle (Nosek et al., 2004).
No pathogenic point mutation has been identified in the N-terminal
variable region of TnT, whereas multiple such mutations have been found
immediately outside the N-terminal variable region (for example I79N of
adult cardiac TnT that causes familial hypertrophic cardiomyopathy
(Knollmann et al., 2001)). This observation may indicate the highly plastic

nature of the N-terminal variable region of TnT.
Nonetheless, larger structural variations such as aberrant splicing in
the N-terminal variable region of cardiac TnT have been reported in


Modulation of Muscle Contractility and Beyond

13

cardiomyopathies. In turkey hearts, abnormal skipping of exon 8 in cardiac
TnT was found in inherited dilated cardiomyopathy (Biesiadecki and Jin,
2002). Counterpart of this exon (exon 7) in mammalian cardiac TnTwas also
spliced out in dog hearts with dilated cardiomyopathy (Biesiadecki et al.,
2002). This exon encodes a normally constitutive segment in cardiac TnT
(Jin et al., 2008; Wei and Jin, 2011). Its aberrant splice-out in dilated cardiomyopathy turkey and dog hearts indicates a causal relationship to the
pathogenesis. Supporting this notion, transgenic mouse studies showed that
overexpression of exon 7-deleted cardiac TnT in adult cardiac muscle
decreased systolic function of the heart (Wei et al., 2010). In addition to
the splice-out of exon 7, the dilated cardiomyopathy dog hearts also had an
abnormal inclusion of the embryonic exon 5 in cardiac TnT in the adult
cardiac muscle (Biesiadecki et al., 2002). The pathophysiologic significance
of embryonic cardiac TnT in adult cardiac muscle will be discussed later.
Alternative splicing of exon 4 that encodes 4–5 amino acids in the Nterminal variable region of cardiac TnT is also related to disease conditions.
Significant expression of low molecular weight cardiac TnTexcluding exon
4 was found in failing human hearts (Anderson et al., 1995; MesnardRouiller et al., 1997), diabetic rat hearts (Akella et al., 1995), and hypertrophic rat hearts (McConnell et al., 1998) (it is worth mentioning that the
abnormally increased exclusion of exon 4 in cardiac TnTwas misinterpreted
and quoted in some cardiology text books as re-expression of fetal cardiac
TnT in failing human hearts).
The alternative splicing-generated decreases in size and negative charge of
the N-terminal variable region of cardiac TnT imply a functional adaptation

to these pathologic conditions. Supporting this hypothesis, the low molecular
weight slow TnT with the exon 5-encoded segment spliced out was upregulated in the muscles of type 1 (demyelination) but not type 2 (axon loss)
Charcot–Marie–Tooth disease, suggesting a functional significance in skeletal
muscle adaptation to neuromuscular disorders (Larsson et al., 2008).
The aberrant splicing of the N-terminal variable region of cardiac TnT
does not abolish the core function of TnTand adult skeletal muscles normally
contain multiple N-terminal alternatively spliced variants of fast and slow
TnT (Jin et al., 2008; Wei and Jin, 2011). Therefore, the mechanism for the
aberrantly spliced cardiac TnT to contribute to the pathogenesis of dilated
cardiomyopathy in turkeys and dogs raised a key question regarding the
structure–function relationship of TnT in cardiac muscle. An important
feature of vertebrate hearts is the synchronized and uniform ventricular
contraction activated as an electrical syncytium. Accordingly, uniform


14

Jian-Ping Jin

TnT function is beneficial for the rhythm pumping function of the heart.
This is different from the function of skeletal muscle, in which multiple TnT
isoforms are present to fit the need of broader twitches for fusion into tetanic
contractions. Based on this observation, we tested a hypothesis that the
abnormality of aberrant N-terminal splicing of cardiac TnT is not a simple
loss of function but the chronic presence of more than one class of TnT in the
thin filaments of adult cardiac muscle (Feng and Jin, 2010).
In this hypothesis, desynchronized activation of ventricular muscle at the
myofilament level due to the coexistence of TnT variants that produce split
Ca2+ sensitivity would decrease the efficiency of cardiac pumping. To demonstrate this mechanism, we first created transgenic mouse hearts that coexpress a wild-type fast skeletal muscle TnT and the endogenous cardiac TnT.
The coexistence of two nonmutant TnT’s in adult cardiac muscle altered the

overall cooperativity of Ca2+-activated force production (Huang et al.,
1999a), decreased cardiac function, and produced myocardial degeneration
(Huang et al., 2008). We then tested in transgenic mouse hearts the effects of
expressing one or two of the cardiac TnT splicing variants found in turkey
and canine-dilated cardiomyopathy together with endogenous wild-type
adult cardiac TnT on cardiac efficiency. The results showed that the coexistence of more than one forms of cardiac TnT in adult cardiac muscle
significantly decreased cardiac pumping efficiency proportional to the
degree of TnT heterogeneity (Feng and Jin, 2010) that splits thin filament
calcium sensitivity (Biesiadecki and Jin, 2002).
It is worth noting that abnormal inclusion of the embryonic exon 5 in
adult cardiac TnT was also found in cat and Guinea pig hearts (Biesiadecki
et al., 2002). In addition, the Guinea pig hearts express cardiac TnTwith an
exclusion of a larger segment in the N-terminal region encoded by exon 6
(Biesiadecki et al., 2002). Cats and Guinea pigs are both reported to have
high incidence of inherited cardiomyopathy and heart failure (Hasenfuss,
1998; Tilley et al., 1977). Therefore, improper splicing of N-terminal exons
of cardiac TnT might be a common pathogenic mechanism.
The alternatively spliced N-terminal coding exons of fast, cardiac, and
slow TnT genes are summarized in Figure 3 and Table 1. The large number
of alternatively spliced TnT variants differing in the N-terminal region may
provide a capacity of modifying muscle contractility whereas retaining the
core functions of TnT.
A very interesting observation is a point mutation of turkey cardiac TnI
(R111C) in the TnI–TnT interface (Biesiadecki et al., 2004), which
blunted the functional effect of protein kinase A phosphorylation of cardiac


15

Modulation of Muscle Contractility and Beyond

N-terminal
variable region

Conserved regions

Tropomyosin-binding site 1

w

Exons 2 3 4 5

X(P)7

y Fetal
67 8
9 10

11

12

TnC-binding site

Tropomyosin-binding site 2 Tnl-binding site

13

14

15

16/17 18

Fast
Chymotrypsin
An additional exon in chicken

(Fetal)

Exons 2 3 4 5

6

7 8

9

10

11

12

8

9

10

13

14

15

16

17

11

12

13

14

Cardiac
Calpain I
Exons 2 3 4 5

6

7

Slow
Alternative acceptor sites in intron 5

Figure 3 Alternatively spliced exons of mammalian and avian fast, cardiac, and slow TnT

genes. The linear maps of fast, cardiac, and slow TnT illustrate the segments encoded by
each exon. The alternatively spliced exons are indicated by the filled boxes, among
which the developmentally regulated exons are in solid black. The w, x (P), and y exons
illustrated in the fast TnT structure are only found in avian species. The alternative
acceptor site involved in the splicing of exon 6 in slow TnT gene is indicated with an
arrowhead. The C-terminal and middle regions of TnT are well conserved among the
three muscle-type-specific isoforms and across species whereas the N-terminal region is
highly variable. The calpain I cleavage site for the selective removal of the N-terminal
variable region of cardiac TnT in stress conditions (Zhang et al., 2006) and the
chymotrypsin cleavage site dividing the T1 and T2 fragments of fast TnT (Perry, 1998)
are indicated with arrowheads.

TnI (Wei et al., 2010) had mutually rescuing effects when it coexists with
the exon 7-deleted cardiac TnT (Biesiadecki et al., 2004; Wei et al., 2010)
in the hearts of double transgenic mice (Wei et al., 2010). This finding
suggests that the TnI–TnT interface is a pivotal site in transmitting Ca2+
signals during striated muscle contraction and relaxation as well as in
mediating the functional effects originating from the N-terminal variable
region of TnT (Jin et al., 2008; Wei and Jin, 2011; Sheng and Jin, 2014).

5. DEVELOPMENTAL REGULATIONS
The expression of TnT isoform genes in embryonic striated muscles
was not as restricted to fiber types as that in the adult animal. Cardiac TnT is
expressed at significant levels in embryonic and neonatal skeletal muscles


16

Jian-Ping Jin

Table 1 Modifications and regulations of the N-terminal variable region of TnT

Exons of TnT genes, which are alternatively spliced under physiologic
conditions
Exons 4, 5, 6, 7, and 8 in avian and mammalian fast TnT
A fetal exon in mammalian fast TnT
P exons (up to 7) in avian fast TnT
Exons w and y in avian fast TnT
Exon 5 in avian and mammalian cardiac TnT
Exon 4 in mammalian cardiac TnT
Exon 5 in mammalian slow TnT
Alternative acceptor sites in intron 5 of mouse slow TnT and intron 4 of chicken
slow TnT
Developmental regulations that result in isoform switches
Alternative splicing of exon 5 in cardiac TnT
Alternative splicing of exons 4, 6, 7, 8, and fetal in fast TnT
Postnatal inclusion of P exons in avian pectoral muscle fast TnT
Posttranslational modifications
Constitutive phosphorylation of Ser2 at the N terminus
Selective removal of the N-terminal variable region by restrictive proteolysis in
adaptation to stress conditions
Functional significance
Variable N-terminal negative charges determine the overall charge of TnT
N-terminal structures modulate the conformation and function of the middle and
C-terminal regions of TnT
N-terminal variation in TnT alters thin filament Ca2+-sensitivity and force
production
N-terminal acidic residues of TnT provide a potential reservior of Ca2+
Deletion of the N-terminal variable region of cardiac TnT moderately decreases
systolic velocity of the heart and increases ejection time and stroke volume

Deletion of the N-terminal variable region of TnT restores an ancestral
conformation
Pathologic alternative splicing
Splice-out of exon 4 in adult cardiac TnT in human heart failure
Splice-out of exon 4 in adult cardiac TnT in diabetic and hypertrophic rat
hearts
Splice-out of exon 8 or exon 7 in cardiac TnT in turkey and dog dilated
cardiomyopathies
Splice-in of exon 5 in adult cardiac TnT in dog dilated cardiomyopathy
Splice-out of exon 6 in cardiac TnT of Guinea pig heart
Splice-out of exon 5 in slow TnT in type 1 Charcot–Marie–Tooth disease
The table summarizes the alternative splicing and posttranslational modifications of the N-terminal
variable region of vertebrate TnT. The relevant developmental regulation, physiologic and pathologic
significances are listed.


Modulation of Muscle Contractility and Beyond

17

(Cooper and Ordahl, 1984; Jin, 1996; Jin et al., 2003; Toyota and Shimada,
1981). Insitu hybridization studies found that the expression of cardiac TnT
in the developing heart begins at day 7.5 postcoitum and in skeletal muscles
at day 11.75 postcoitum (Wang et al., 2001). The expression of cardiac TnT
gene is downregulated in skeletal muscles during postnatal development and
ceases in the adult (Jin et al., 2003; Sabry and Dhoot, 1991; Saggin et al.,
1990). The developmental switching from cardiac TnT to skeletal muscle
TnT is seen in both avian and mammalian skeletal muscles (Cooper and
Ordahl, 1984; Jin, 1996; Swiderski and Solursh, 1990; Toyota and Shimada,
1981), demonstrating a functional exchangeability between the muscle typespecific TnT isoforms. On the other hand, the developmentally regulated

switch of TnT isoforms indicates differentiated function of the TnT isoforms
in different types of adult striated muscles.
While the expression of cardiac TnT gene is downregulated, the expression of slow TnT is upregulated in postnatal slow skeletal muscles. This
process is concurrent with the onset of the Amish nemaline myopathy in
which the affected infant’s lack of slow TnT in their skeletal muscle are
apparently normal in skeletal muscle function at birth but soon develop the
disease phenotypes while cardiac TnT ceases expression in skeletal muscles
(Jin et al., 2003). This observation suggests that cardiac TnT may function in
place of slow TnT in embryonic and growing skeletal muscles, a hypothesis
that is worth testing for the development of targeted therapeutic approaches
of Amish nemaline myopathy.
Transient expression of slow TnT, but not fast TnT, was found in the
embryonic heart. At day 13.5 postcoitum, expressions of all three TnT genes
were detected in the developing tongue and this coexpression continued to
day 16.5 postcoitum with fast TnT being predominant. Cardiac TnT transcript was also detectable by in situ hybridization in the embryonic urinary
bladder, where presumably smooth muscle was present (Wang et al., 2001). It
remains to be investigated whether this low-level expression of TnT in
smooth muscle has a physiologic significance.
In chicken skeletal muscle, cardiac TnC was coexpressed with cardiac
TnT in early developmental stages (Toyota and Shimada, 1981). During the
development of avian skeletal muscle, the downregulation of cardiac TnT
and cardiac TnC and the upregulation of the adult form of skeletal troponin
subunits were dependent on diffusible neurohumoral factors but independent of functional innervation (Toyota and Shimada, 1983).
As discussed earlier, the alternative splicing of cardiac TnT switches
pattern during avian and mammalian heart development. Embryonic and


18

Jian-Ping Jin

neonatal hearts express embryonic cardiac TnTwith the inclusion of a 9 or 10
amino acid segment encoded by exon 5 in the N-terminal region. The
embryonic cardiac TnT is later replaced with adult cardiac TnT by excluding
the exon 5-encoded segment (Cooper and Ordahl, 1985; Jin et al., 1992,
1996; Jin and Lin, 1989). The exon 5-encoded segment is highly acidic
(negatively charged at physiologic pH) and, therefore, this alternative splicing
regulation represents a large to small and more acidic to less acidic transition
of the physical properties of cardiac TnT (Jin and Lin, 1988). The time course
of this developmental switch has been described for chicken, mouse, and rat
hearts. Cardiac TnT cDNAs with the same embryonic and adult splicing
patterns are also found in human hearts (Townsend et al., 1995) and the same
protein isoform switch was seen in developing human skeletal muscles where
cardiac TnT is transiently expressed (Jin et al., 2003).
Complex alternative splicing of fast skeletal muscle TnT occurs during
skeletal muscle development involving multiple coding exons for the
N-terminal variable region. Similar to that of cardiac TnT, a fetal exon
located between the alternative exon 8 and constitutive exon 9 is found in
mammalian fast TnT genes (Briggs and Schachat, 1993). Inclusion of the
fetal exon-encoded segment in the embryonic fast TnT had an inhibitory
effect on myosin ATPase activity in reconstituted myofilaments (Chaudhuri
et al., 2005). Involving the fetal exon and multiple other N-terminal alternative exons (exons 4, 6, 7, and 8) encoding mainly acidic residues, the
expression of fast TnT also exhibits a developmental switching from high
molecular weight acidic isoforms to low molecular weight basic isoforms
(Wang and Jin, 1997).
Whereas, most of the N-terminal alternatively spliced exons of fast and
cardiac TnT genes exhibit decreased inclusion during heart and skeletal
muscle development (Jin et al., 1996; Sheng and Jin, 2014; Wang and Jin,
1997; Wei and Jin, 2011), a unique case in the developmental regulation of
fast TnT gene is the posthatching inclusion of seven P exons encoding the Tx

segment in the N-terminal region of avian pectoral but not leg muscles (Jin
and Samanez, 2001; Ogut and Jin, 1998). While the large number of Glu
residues encoded by the P exons might serve as a calcium reservoir in avian
pectoral muscle thin filaments (Zhang et al., 2004) and the negative charges
added to the N-terminal variable region of Tx-positive fast TnT correlated
to an increased tolerance to acidosis (Ogut and Jin, 1998), the biologic
significance of the Tx segment and its developmentally regulated expression
in adult avian pectoral muscles, especially its capacity of binding transition
metal ions, remains to be investigated.


Modulation of Muscle Contractility and Beyond

19

The developmentally regulated alternative N-terminal coding exons of
the three muscle fiber type TnT genes are summarized in Table 1. The
N-terminal variable region confers the most significant difference between
the embryonic and adult isoforms and plays a role in modulating the overall
conformation of TnTand the interactions with TnI, TnC, and tropomyosin.
Altogether, the developmental regulation of TnT gene expression and alternative splicing provides adaptive modifications for the contractility of cardiac
and skeletal muscles (Jin et al., 2008; Wei and Jin, 2011).
The cellular mechanism(s) that regulates TnT alternative splicing during
development remains to be established. When cardiac TnT is naturally
expressed in embryonic and neonatal skeletal muscles, its splicing pattern
is synchronized with the developmental switching in the heart (Jin, 1996).
This observation indicates the role of a systemic biological clock independent of the very different functional adaptations during the postnatal development of cardiac and skeletal muscles.
More recent studies demonstrated that microRNAs play a role in regulating striated muscle development and pathophysiologic remodeling
(Tatsuguchi et al., 2007; van Rooij et al., 2009; Williams et al., 2009). In
adult mouse heart, the deletion of miR-208a increased the expressions of fast

TnI and fast TnT, which could be corrected by overexpression of miR-499
(van Rooij et al., 2009). In skeletal muscle, double deletion of miR-208b and
miR-499 lead to decreased number of slow fibers (van Rooij et al., 2009).

6. POSTTRANSLATIONAL MODIFICATIONS
Posttranslational modification of proteins provides rapid functional
regulations. The posttranslational regulation of TnT has been mainly investigated for the roles of phosphorylation and restricted proteolysis. In contrast
to the chronic mechanisms of TnT isoform gene regulation and alternative
RNA splicing, the modification of TnT structure through phosphorylation
and restricted proteolysis are acute mechanisms for the muscle to adapt to
functional demands and stress conditions.

6.1 Phosphorylation
Various in vitro and ex vivo experimental conditions produced phosphorylation of cardiac TnTat multiple sites. For example, Thr197, Ser201, Thr206, and
Thr287 in the C-terminal region of cardiac TnTwere reported to be protein