TNNT1, TNNT2, and TNNT3: Isoform Genes, Regulation, and Structure-Function Relationships

Abstract

Troponin T (TnT) is a central player in the calcium regulation of actin thin filament function and is essential for the contraction of striated muscles. Three homologous genes have evolved in vertebrates to encode three muscle type-specific TnT isoforms: TNNT1 for slow skeletal muscle TnT, TNNT2 for cardiac muscle TnT, and TNNT3 for fast skeletal muscle TnT. Alternative splicing and posttranslational modifications confer additional structural and functional variations of TnT during development and muscle adaptation to various physiological and pathological conditions. This review focuses on the TnT isoform genes and their molecular evolution, alternative splicing, developmental regulation, structure-function relationships of TnT proteins, posttranslational modifications, and myopathic mutations and abnormal splicing. The goal is to provide a concise summary of the current knowledge and some perspectives for future research and translational applications.

Graphical Abstract

1. Introduction

The basic contractile unit of vertebrate striated muscle, i.e., skeletal and cardiac muscles, is the sarcomeres in myofibrils, which consist of overlapping myosin thick filaments and actin thin myofilaments (Tobacman 1996; Gordon, Homsher et al. 2000). Muscle contraction is powered by actin-activated myosin II motor (Berchtold, Brinkmeier et al. 2000), which converts the energy from ATP hydrolysis to a power stroke of myosin head during each cross-bridge cycle. This process is regulated by calcium-mediated protein conformational changes in both thick and thin myofilament of the sarcomere (Tobacman 1996).

The troponin complex associated with the actin thin filament plays a central role in the regulation of muscle contraction and relaxation. The troponin complex has three protein subunits: The calcium binding subunit troponin C (TnC) (Chaudhuri, Mukherjea et al. 2005), the actomyosin ATPase inhibitory subunit troponin I (TnI), and the tropomyosin binding and thin filament anchoring subunit troponin T (TnT). Troponin T interacts with TnI, TnC, tropomyosin and actin to play an organizer role in the calcium regulation of muscle contraction and relaxation (Jin, Zhang et al. 2008).

Discovered in the 1960s (Ebashi 1963; Kato, Negishi et al. 1969), vertebrate TnT is a 30~35kDa myofilament protein. Visualized by electron microscopy, TnT anchors troponin complex on the actin-tropomyosin thin filament at ~37 nm intervals (Otsuki, Masaki et al. 1967). The stoichiometry of TnT binding to F-actin is in a ratio of 1 (TnT) to 7 (actin monomers) (al-Hillawi, Bhandari et al. 1995). During the last two decades, extensive studies revealed that vertebrate TnT is encoded by three homologous genes that specifically expressed in slow skeletal muscle (TNNT1), cardiac muscle (TNNT2), and fast skeletal muscle (TNNT3) (Barton, Townsend et al. 1997) (Table 1).

Table 1.

Human TnT isoform genes and tissue-specific expression

Protein isoforms	Slow Skeletal Muscle TnT	Cardiac TnT	Fast skeletal Muscle TnT
Gene	TNNT1	TNNT2	TNNT3
Chromosomal Location	19q13.4	1q32	11p15.5
Number of Exons	14	17	19
Number of Amino Acids	261	297	268
Relative Molecular Mass	32948.15	35923.49	30596.19
Isoelectric Point	5.95	4.88	6.21
Tissue Specificity	Slow-twitch fibers	Heart Embryonic skeletal muscle	Fast-twitch fibers

Molecular mass and isoelectric point of human TnT isoforms were calculated using the following protein sequences: TNNT1, NM_003283; TNNT2, NM_001276345; and TNNT3, NM_006757.

Early protein sequencing work and mainly later cDNA cloning studies have determined the primary structure of TnT isoforms in multiple species (Jin, Zhang et al. 2008). Due to its extended molecular conformational and allosteric structure (Cabral-Lilly, Tobacman et al. 1997; Wendt, Guenebaut et al. 1997), crystallographic data for the troponin complex only determined the high resolution structure of a C-terminal portion of TnT (Takeda, Yamashita et al. 2003; Vinogradova, Stone et al. 2005), whereas the N-terminal and middle regions and the last 14 amino acids at the C-terminal end of TnT were missing. Therefore, most of the current understanding of TnT structure and its interaction with other thin filament proteins are based on protein binding studies and conformational changes detected using monoclonal antibody (mAb) epitope analysis and fluorescence spectral study.

In addition to anchoring the troponin complex on the thin filament, TnT dynamically interacts with other thin filament regulatory proteins to conduct the calcium signaled excitation-contraction-relaxation cycles of striated muscle (Jin, Zhang et al. 2008). The present review focuses on the molecular evolution of TnT isoform genes, alternative splicing, developmental regulation, posttranslational modifications, structure-function relationship of TnT protein, myopathic mutations, and splicing abnormalities. More detailed information can be found in several previous review articles (Tobacman 1996; Perry 1998; Gordon, Homsher et al. 2000; Jin, Zhang et al. 2008; Sheng and Jin 2014).

2. Molecular evolution of TNNT genes encoding muscle type-specific TnT isoforms

Three homologous genes have evolved in vertebrates, encoding the cardiac (TNNT2), slow skeletal muscle (TNNT1) and fast skeletal muscle (TNNT3) isoforms of TnT. The three TnT isoforms are significantly diverged in the N-terminal region but highly conserved in the middle and C-terminal regions that contain binding sites for TnC, TnI and tropomyosin (Figure 1) (Jin, Zhang et al. 2008). While TNNT1 and TNNT3 are specifically expressed in slow and fast twitch skeletal muscle fibers, respectively, TNNT2 is expressed in cardiac muscle and transiently expressed in embryonic and neonatal skeletal muscles, including both slow and fast fiber dominant muscles, in avian and mammalian species (Toyota and Shimada 1983; Cooper and Ordahl 1985; Jin 1996).

Figure 1. Structural and functional domains and alternatively spliced exons of the three TnT isoforms.

A. Partial crystallography structure of troponin complex was modified from published data of cardiac troponin (Takeda, Yamashita et al. 2003) using UCSF chimera software. The calpain cleavage site, caspase 3 cleavage sit, chymotryptic cleavage site between TnT T1 and T2 fragments, and the two tropomyosin-binding sites are indicated. B. The linear structure alignment shows that the N-terminal region of TnT is highly variable in cardiac, fast and slow skeletal muscle isoforms and regulated via alternative splicing, whereas the middle and C-terminal regions are highly conserved. The filled boxes indicate alternative spliced exons, of which the solid boxes represent those under developmental regulation. The alternatively spliced exons w, x (P) and y in the fast TnT exon map are only found in avian species. The T1 and T2 fragments generated by limited chymotryptic digestion are outlined. The calpain and caspase cleavage sites in cardiac TnT are also indicated.

Knockout of mouse cardiac TnT gene caused embryonic lethality (Nishii, Morimoto et al. 2008), indicating the lack of compensatory expression of slow or fast skeletal muscle TnT gene in cardiac muscle. Similarly, mutations in TNNT1 gene resulting a complete loss of slow TnT in slow skeletal muscle also causes severe nemaline myopathy with childhood lethality (Johnston, Kelley et al. 2000; Jin, Brotto et al. 2003). Therefore, the muscle type-specific TnT isoform genes are non-redundant and differentially expressed in specific type of muscle cells to function in differentiated cellular environments.

While the expression of TNNT1 is slow skeletal muscle specific in almost all vertebrate species studied, the heart muscle of dry land toad (Bufo) utilizes exclusively slow TnT other than cardiac TnT (Feng, Chen et al. 2012). Multiple representative vertebrate species studied from fish to human, including the closely related frog, all express only cardiac TnT in the cardiac muscle. Therefore, the unique utilization of slow TnT in toad heart reflects a unique evolutionary selection, potentially with a value in the adaptation of cardiac function to the dry land habitats of toad, which causes drastic change in blood volume and stress on cardiac function. Supporting this observation, studies of cardiac function have showed that toad heart is significantly more tolerant to increases in afterload than that of frog heart (Feng, Chen et al. 2012).

Troponin T and TnI genes are located in vertebrate genomes in three closely linked pairs: fast TnI-fast TnT, cardiac TnI-slow TnT and slow TnI-cardiac TnT (Chong and Jin 2009; Wei and Jin 2011). Although TnT and TnI are significantly diverged proteins, their close linkage in the vertebrate genomes and protein epitope analysis suggested that TnT and TnI genes have originated from a TnI-like ancestor gene via gene duplication (Figure 2) (Chong and Jin 2009). Structural integration may have preserved the close chromosomal linkage between the paired TnI and TnT genes. For example, deletion of the genomic DNA segment containing cardiac TnI gene also deleted a part of the slow TnT gene promoter and caused decreased transcriptional activity (Huang, Chen et al. 1999) and expression of slow TnT in slow muscle fibers (Feng, Wei et al. 2009).

Figure 2. Molecular evolution lineage of vertebrate TnT isoform genes.

Modified from Chong and Jin (2009), the illustration shows the evolutionary lineage of the three closely linked pairs of muscle type specific TnT and TnI genes. Genes encoding TnT and TnI were likely originated from a TnI-like ancestor gene. A fsTnI-like-fsTnT-like gene pair was first emerged by duplication and diversified from the original TnI-like ancestor gene and further gave rise to a ssTnI-like-cTnT-like gene pair. Further duplication and diversification of the cTnI like-cTnT-like gene pair occurred as a later event form the three present-day muscle type specific pairs of TnT and TnI isoform genes in vertebrates.

The cardiac TnI-slow TnT and slow TnI-cardiac TnT gene pairs are apparently not linked in a fiber type-specific manner. However, slow TnI is expressed as the sole TnI together with cardiac TnT in embryonic heart (Saggin, Ausoni et al. 1988; Saggin, Gorza et al. 1989). Therefore, the slow TnI-cardiac TnT gene pair is actually a functionally linked pair. It remains to be investigated whether the cardiac TnI-slow TnT gene pair also has a functional significance.

Sequence analysis and protein epitope studies further revealed the evolutionary linage of the three TnT isoform genes (Figure 2) (Chong and Jin 2009). A novel approach was used to detect ancestor conformations that have been repressed allosterically in the three dimensional structures in present-day TnT proteins by the addition of later modulator structures during evolutionary diversification. These modulator structures served as evolutionary determinants for their structural effect on repressing the ancestral conformation (Chong and Jin 2009). For example, the N-terminal variable region of TnT has been identified as a repressive modulator. Removing this conformational modulator peptide fragment of TnT restored epitopic structures of the origin of an ancestral TnT isoform, which are detectable using mAb probes. The epitope analysis revealed the evolutionary lineage relationships between the muscle fiber type-specific TnT isoform genes as well as between TnI and TnT genes.

Experimental data demonstrated that a fast TnI-like-fast TnT-like gene pair was first emerged from duplication of a TnI-like ancestor gene (Figure 2). The present-day fast TnI-fast TnT gene pair is derived directly from this ancestor gene pair. A slow TnI-like-cardiac TnT-like gene pair emerged by duplication of the original fast TnI-like-fast TnT-like gene pair. The emergence of cardiac TnI-slow TnT gene pair occurred latest from duplication of the slow TnI-like-cardiac TnT-like gene pair (Chong and Jin 2009). The molecular evolution lineage of TnT-TnI gene pairs provides valuable information for understanding the structure-function relationships of TnT.

Studies of primary structures of numerous TnT protein isoforms revealed that each of the muscle type specific TnT isoforms in different vertebrate species is more conserved than the three muscle type specific TnT isoforms in a given species (Figure 3) (Jin, Zhang et al. 2008). This observation suggests that the demands for muscle type specific functional adaptations in cardiac, slow and fast muscles provided the primary selection pressure and have driven the diversification and conservation of TnT isoform genes during evolution (Chong and Jin 2009).

Figure 3. Phylogenic tree of vertebrate TnT isoforms.

A phylogenetic tree of vertebrate Tnnt genes is derived from protein sequence alignment performed with the Clustal V method using the MegAlign computer program (Lasergene; DNASTAR, lnc, Madison, WI). The degree of divergence is indicated with the length of the lineage lines. Tnnt1, 2 and 3 isoforms are marked in blue, red and green, respectively. The NCBI database accession numbers for the sequences analyzed are: African clawed frog Tnnt1, NP_001086207.1; African clawed frog Tnnt2, AAO33405.1; African clawed frog Tnnt3, NP_001080403.1; Atlantic cod Tnnt3, AAM21701.1; baboon Tnnt3, NP_001162538.1; bat Tnnt1, ELK09186.1; bat Tnnt3, ELK13205.1; blunt snout bream Tnnt2, bovine Tnnt1, NP_776899.1; bovine Tnnt2, NP_777196.1; bovine Tnnt3, BAB92975.1; buzzard Tnnt2, KFP58299.1; cat Tnnt2, AAO13362.1; channel fish Tnnt1, AHH42303.1; channel fish Tnnt3, AHH42694.1; chicken Tnnt1, BAD06455.1; chicken Tnnt2, NP_990780.1; chicken Tnnt3, NP_990253.1; chimpanzee Tnnt1, JAA42790.1; chimpanzee Tnnt2, JAA36029.1; chimpanzee Tnnt3, JAA19567.1; Chinese hamster Tnnt2, EGW02189.1; Chinese tree shrew Tnnt3, ELW71918.1; Cormorant Tnnt2, KFW92284.1; crestless curassow Tnnt3, AAF81014.1; dog Tnnt2, NP_001003012.2; ghost shark Tnnt2, AFP07845.1; ghost shark Tnnt3, AFP05162.1; goat Tnnt2, NP_001301119.1; goat Tnnt3, NP_001301139.1; Guinea pig Tnnt2, NM_001172863.1; human TNNT1, CAA09752.1; human TNNT2, AAK92231.1; human TNNT3, AAF21629.1; large yellow croaker Tnnt1, KKF32449.1; mouse Tnnt1, AAD00730.1; mouse Tnnt2, AAA85350.1; mouse Tnnt3, AAF01502.1; naked mole rat Tnnt1, EHB13469.1; pig Tnnt1, NP_998913.1; pig Tnnt2, NP_001244282.1; pig Tnnt3, NP_001001863.1; pigeon Tnnt3, EMC88041.1; rabbit Tnnt3, GU944669.1; rat Tnnt1, NM_134388.2; rat Tnnt2, NP_036808.1; rat Tnnt3, NM_001270665.1; Rhesus monkey Tnnt2, NM_001247991.1; salmon Tnnt1, ACM09392.1; salmon Tnnt2, NP_001140134.1; salmon Tnnt3, ACM08284.1; sun bittern Tnnt2, KFW09657.1; toad Tnnt1, AY773671.2; turkey Tnnt2, NM_001303165.1; turkey Tnnt3, NP_001290143.1; turtle Tnnt2, EMP40998.1; turtle Tnnt3, EMP32847.1; KC556826.1; white-tailed tropicbird Tnnt2, KFQ82867.1; western clawed frog Tnnt3, NP_989143.1; zebra fish Tnnt1, NP_001122167.1; zebra fish Tnnt2, AAL06279.1; zebra fish Tnnt3, AAF78472.1.

3. Structure-function relationships of TnT

Troponin T is a protein of 30~35kDa in size containing ~220 to 300 amino acids. Amino acid sequence comparison demonstrated that the diversity of TnT isoforms is largely in the N-terminal variable region, whereas the middle and C-terminal regions are highly conserved (Jin, Zhang et al. 2008). The structure-function relationship of TnT has been extensively investigated in protein binding studies using TnT fragments (Figure 1). The chymotryptic fragments T1 and T2 of rabbit fast skeletal TnT (Tanokura, Tawada et al. 1981) were studied for their interactions with other regulatory proteins in the sarcomeric thin filament. The T1 fragment binds to the head-to-tail junction of tropomyosin and the T2 fragment binds to the middle region of tropomyosin and also binds to TnI in the troponin complex (Pato, Mak et al. 1981; Tanokura, Tawada et al. 1983; Tanokura and Ohtsuki 1984; White, Cohen et al. 1987).

High resolution X-ray crystallographic structures have been obtained from human cardiac troponin complex and chicken skeletal muscle troponin complex. The high resolution data revealed only a small portion of the TnT structure but included the regions interacting with TnI and TnC (Takeda, Yamashita et al. 2003; Vinogradova, Stone et al. 2005). Troponin T binds TnI through a helical coiled-coil interface corresponding to Leu₂₂₄-Val₂₇₄ of human cardiac TnT and Glu₁₉₉-Gln₂₄₅ of chicken fast skeletal TnT. This interface structure is referred to as the I-T arm region. Amino acid sequences within the I-T arm region are highly conserved in the three muscle type specific TnT isoforms (Jin, Zhang et al. 2008). The 12 amino acids at the very C-terminal end of TnT were missing from the crystal structures (Figure 1). This segment is conserved in vertebrate TnT and its function remains to be investigated.

Not resolved in the high resolution crystal structures, two tropomyosin binding sites of TnT have been identified by protein binding studies. A T1 region tropomyosin binding site has been mapped to a 39-amino acid segment at the N-terminal portion of the middle conserved region of TnT. A tropomyosin binding site in the T2 fragment was mapped to a 25-amino acid segment at the beginning of the T2 fragment (Jin and Chong 2010). Amino acid sequences of the segments containing these tropomyosin binding sites are also highly conserved in the three muscle type specific TnT isoforms and across vertebrate species (Jin, Zhang et al. 2008).

The structure of the N-terminal region of TnT is hypervariable in different muscle types and regulated during development. The CNBr fragment CB3 of rabbit fast skeletal muscle TnT (amino acids 2–50) representing the N-terminal variable region does not bind any known myofilament proteins (Pearlstone and Smillie 1982; Heeley, Golosinska et al. 1987). More recent studies have demonstrated a role of the N-terminal variable region in the regulation of TnT molecular conformation and interaction with other myofilament regulatory proteins.

Monoclonal antibody epitope analysis demonstrated that the binding of metal ions or structural variations in the N-terminal region alters epitope conformation in the middle and C-terminal regions (Wang and Jin 1998; Jin, Chen et al. 2000). This mechanism was confirmed by spectral analysis of fluorescence of residues in the C-terminal region (Jin and Root 2000). Such modulatory effects reflect that the N-terminal region regulates the molecular conformation of TnT.

The N-terminal variable region based regulation results in functional alterations. Microplate protein binding assays showed that Zn²⁺ binding to the N-terminal variable region of chicken fast skeletal TnT decreased the binding affinity of TnT for tropomyosin (Ogut and Jin 1996; Wang and Jin 1998). Protein binding studies further demonstrated that the N-terminal variable region of TnT modulates the binding affinity of TnT for tropomyosin primarily by reducing the affinity of site 1 in the middle region (Amarasinghe and Jin 2015). Physiological significance of the regulatory effects of the N-terminal variable region of TnT have also been demonstrated in vivo in studies using transgenic mice expressing N-terminal modified cardiac TnT in the heart (Biesiadecki, Elder et al. 2002; Feng, Biesiadecki et al. 2008; Wei, Gao et al. 2010).

In vitro studies using reconstituted troponin complex showed that the presence of the N-terminal fragment in TnT potentiated actomyosin ATPase activity (Malnic, Farah et al. 1998). Physiological studies using transgenic mice expressing cardiac TnT with a deletion of the N-terminal 71 amino acids showed moderately decreased contractile velocity but preserved physiological cardiac function and sustained cardiac output against increases of afterload (Feng, Biesiadecki et al. 2008).

These data demonstrate that the diverged N-terminal structure of the muscle type specific TnT isoforms provides a regulatory mechanism to fine tune the function of troponin and muscle contractility, and reflects the differentiated physiological features of the differentiated muscle fiber types. On the other hand, the conserved structures in the middle and C-terminal regions of TnT reflect the similar baseline function of troponin in the calcium regulation of cardiac, slow and fast skeletal muscle contractions.

4. Developmental regulation of TnT isoform genes and alternative pre-mRNA spicing

Expression of the three TnT isoform genes is regulated during development at the transcriptional level as well as via alternative splicing. Slow and fast skeletal muscle TnT genes have their specific expression primarily in slow and fast twitch skeletal muscle fibers, respectively, throughout development. Cardiac TnT gene is the sole TnT isoform expressed in cardiac muscle, and is expressed at significant levels in embryonic skeletal muscle (Jin 1996). Cardiac TnT is also found in extraocular muscles of adult dog and rat (Bicer and Reiser 2013), further indicating its expression in adult animals is not restricted to the heart.

Alternative splicing plays a major role in generating further diversity in TnT structures for fine tuning cardiac and skeletal muscle contractility during development with changes in functional demands and in adaption to pathological conditions. Alternative splicing of TnT pre-mRNA increases the N-terminal variation in all three TnT isoforms, and generates a C-terminal variable region in fast skeletal muscle TnT and avian cardiac TnT (Jin, Zhang et al. 2008; Wei and Jin 2011). With one exception in avian fast skeletal muscle TnT (to be discussed in the following section), a common feature of TnT N-terminal alternative splicing during embryonic and postnatal development is the switch from high molecular weight to low molecular weight and acidic to basic splice forms (Wei and Jin 2011).

4.1. TNNT3

Mammalian TNNT3 gene encoding the fast skeletal muscle isoform of TnT (fsTnT) contains 19 exons. Alternative TNNT3 pre-mRNA splicing has been observed very early on from the diversity of fsTnT proteins (Wilkinson, Moir et al. 1984). N-terminal coding exons 4, 5, 6, 7 and 8 are alternatively spliced (Wilkinson, Moir et al. 1984; Breitbart and Nadal-Ginard 1986; Wang and Jin 1997). A fetal exon between exon 8 and 9 is also alternatively spliced and exclusively expressed in embryonic fast muscles (Briggs and Schachat 1993). Exons 16 and 17 in the C-terminal region of fsTnT, previously designated as α and β exons respectively, are alternatively spliced in a mutually exclusive manner (Medford, Nguyen et al. 1984).

Avian fsTnT gene has more complex alternative splicing patterns due to the additional alternative exons w, P1-7(x) and y in the N-terminal region (Smillie, Golosinska et al. 1988; Miyazaki, Jozaki et al. 1999; Jin and Samanez 2001). Correspondingly, two-dimensional electrophoresis study of chicken leg muscle protein detected more than 40 different fsTnT protein splice forms (Imai, Hirai et al. 1986).

Expression of TNNT3 during embryonic and postnatal development undergoes a high molecular weight to low molecular weight splice form switch in both fast and slow fiber-dominant skeletal muscles (Wei, Lu et al. 2014). This switch also represents a transition of low isoelectric point splice forms to high isoelectric point forms (Jin, Zhang et al. 2008). The size and charge changes are produced by alternative inclusions of N-terminal exons that encode mainly acidic amino acids (Wang and Jin 1998).

Alternative splicing of the two mutually exclusive C-terminal exons 16 and 17 is also regulated during development (Wang and Jin 1997). Analysis of cDNA sequences suggested that Exon 17 that has a higher similarity in amino acid sequence to its counterparts in slow skeletal muscle TnT and cardiac TnT is predominantly expressed in embryonic and neonatal fsTnT isoforms and exon 16 was mainly found in adult fsTnT (Wang and Jin 1997; Jin, Chen et al. 1998). Exons 16 and 17 both encode a 14 amino acids peptide fragment, which resides in the interface with TnI and TnC (Wang and Jin 1997). Protein interaction studies revealed that incorporation of exon 17 weakened binding of fsTnT to TnC and tropomyosin (Wu, Jha et al. 1995).

Additional alternative N-terminal coding exons have been found in avian Tnnt3 gene with unique splicing patterns (Ogut and Jin 1998). In addition to the high molecular weight to low molecular weight switch via combinations of alternative splicing of exons 4–8 as that in the expression of mammalian fsTnT during development, the expression of a Tx segment encoded by the seven P exons located between exon 5 and 6 in the N-terminal variable region of avian fsTnT (Jin and Smillie 1994) is up-regulated in pectoral but not leg muscles during post-hatch development (Ogut and Jin 1998). Around 28 days after hatch, splice-in of the Tx segment is rapidly up-regulated to become predominant in fsTnT expressed in pectoral muscles of adult birds (Jin and Samanez 2001). The P exons in Tnnt3 gene of avian species in the orders of Galliformes and Craciformes have evolved to encode repeating pentapeptide motifs of AHH(A/E)A. An intriguing finding is that the inclusion of 7 P exons in chicken Tnnt3 mRNA encodes a Tx segment containing seven repeating H(A/E)AAH motifs in the N-terminal variable region of fsTnT, which bind transition metal ions Cu(II), Ni(II), Zn(II) and Co(II) (Jin and Smillie 1994).

The Tx segment is negatively charged and its inclusion cancels the developmental size and charge switch produced by various exclusions of exon 4–8. This feature corresponds to a higher tolerance of adult pectoral muscle fsTnT to acidosis (Ogut and Jin 1998; Jin, Zhang et al. 2008). While the physiological function of the high affinity binding to transition metal ions remains to be investigated, the Tx segment of avian fsTnT shows binding to calcium, potentially serves as a calcium reservoir in avian pectoral muscle myofilaments (Zhang, Jin et al. 2004). Supporting this hypothesis, adult chicken breast muscle exhibits higher calcium sensitivity in myofilament force production than that of Tx-negative muscle (Ogut, Granzier et al. 1999).

Deficiency of slow skeletal TnT did not affect the developmental switch of fsTnT splice forms, indicating that the alternative splicing of TNNT3 pre-mRNA is regulated independent of skeletal muscle fiber types (Wei, Lu et al. 2014). Although the N-terminal variable region of TnT does not bind to any know proteins in the thin filament regulatory system, alternative splicing in the N-terminal segment of fsTnT has shown effects on the overall conformation of TnT and the binding affinities for TnI and tropomyosin (Biesiadecki, Chong et al. 2007).

4.2. TNNT2

Mammalian cardiac TnT gene contains 17 exons, of which 3 are alternatively spliced (Jin, Huang et al. 1992). Exon 13 between the conserved middle and C-terminal regions encodes 2–3 amino acids and is alternatively spliced with unknown function and regulation. Exon 4 in the N-terminal region is alternatively spliced in both adult and embryonic hearts (Jin, Wang et al. 1996; Farza, Townsend et al. 1998), of which the functional significance also remains to be investigated.

Exon 5 in the N-terminal region of cardiac TnT is expressed in embryonic heart and excluded from adult cardiac TnT (Jin and Lin 1989). Exon 5 of TNNT2 gene encodes a 9 or 10 amino acids segment that is highly acidic and negatively charged at physiological pH (Jin, Zhang et al. 2008). Embryonic cardiac TnT with more negative charges in the N-terminal region exerts higher calcium sensitivity of actomyosin ATPase activity and myofilament force production (Gomes and Potter 2004) as compared with that of adult cardiac TnT. This function may also correspond to the effect of TnT isoforms on the tolerance of cardiac muscle to acidosis (Nosek et al., 2004).

When TNNT2 is transiently expressed in embryonic and neonatal skeletal muscles, its alternative splicing pattern is synchronized to that in the heart (Jin 1996). While the isoform switch from slow to cardiac TnI regulated at the transcriptional level corresponds to functional demands during postnatal heart development, timing of the switching of TNNT2 alternative splicing may vary in different species (Jin 1996), indicating the regulation by a genetically programmed biological clock, rather than adaptation to changes in contractile function.

4.3. TNNT1

The expression of slow skeletal muscle TnT gene involves relatively less complex alternative splicing in comparison to that of cardiac and fast skeletal muscle TnT genes. Among the 14 exons of TNNT1, exon 5 encoding an 11-amino acid segment in the N-terminal region is alternatively spliced, generating a high molecular weight and a low molecular weight slow TnT variants (Gahlmann, Troutt et al. 1987; Jin, Chen et al. 1998; Huang, Chen et al. 1999). In addition, splicing at alternative acceptor sites of TNNT1 pre-mRNA produces a single amino acid difference in the peptide segment encoded by exon 6 (Huang, Chen et al. 1999). The alternative splicing of slow skeletal muscle TnT pre-mRNA has not been found with any correlation to muscle development.

Discussed above, the heart muscle of dry land toad utilizes exclusively slow TnT other than cardiac TnT reflecting a unique evolutionary selection with a value in the adaptation of cardiac function to the drastic changes in blood volume (Feng, Chen et al. 2012). Interestingly, the slow TnT expressed in toad heart is the low molecular weight splice form excluding the exon 5-encoded segment (Feng, Chen et al. 2012). The low molecular weight slow skeletal muscle TnT is also predominantly expressed in overused prior polio muscle and up-regulated in type 1 demyelination, but not type 2, Charcot-Marie-Tooth disease (CMT) (Larsson, Wang et al. 2008). Based on the observation that the N-terminal structural variations alter the molecular conformation and function of the middle and C-terminal regions with functional effects on the interactions with TnI, TnC and tropomyosin, the alternative splicing regulation of TNNT1 expression may play an important role in modulating muscle contractility in physiological and pathophysiological adaptations.

5. Posttranslational modifications

Posttranslational modifications can rapidly regulate the function of proteins in physiological and pathophysiological conditions. Known posttranslational modifications of TnT include phosphorylation, O-linked GlcNAcylation and proteolytic modifications. Among the three TnT isoforms, posttranslational modifications are most intensely studied in cardiac TnT with an emphasis on the significance in heart diseases.

5.1. Phosphorylation

Phosphorylation is a fundamental mechanism in the regulation of the structure and function of myofilament proteins, including TnT. In both cardiac and skeletal muscle cells, TnT is phosphorylated at multiple Ser and Thr residues. Table 2 summarizes major phosphorylation sites of cardiac TnT and their functional effects on the contractility and pathophysiological adaptation of cardiac muscle.

Table 2.

Phosphorylation sites in TnT

Phosphorylation site			Kinases	Function	Reference
cTnT	ssTnT	fsTnT
Ser2	C	C	Unknown	Unknown	(Villar-Palasi and Kumon 1981; Gusev, Barskaya et al. 1983; Zhang, Zhang et al. 2011)
Thr204	N	N	PKC	Reduce Myosin ATPase activity, myofilament force production and Ca2+ sensitivity	(Noland, Raynor et al. 1989; Jideama, Noland et al. 1996; Montgomery, Chandra et al. 2001; Sumandea, Pyle et al. 2003)
Thr204	N	N	CaMK II	Unknown	(Jaquet, Fukunaga et al. 1995)
Thr204	N	N	ASK I	Reduce cardiomyocyte contractility	(He, Liu et al. 2003)
Ser208	N	N	PKC	Reduce Myosin ATPase activity, alter myofilament Ca2+ sensitivity	(Jideama, Noland et al. 1996; Montgomery, Chandra et al. 2001; Sumandea, Vahebi et al. 2009)
Ser208	N	N	ASK I	Reduce cardiomyocyte contractility	(He, Liu et al. 2003)
Thr213	C	C	PKC	Reduce Myosin ATPase activity, myofilament force production and Ca2+ sensitivity	(Noland, Raynor et al. 1989; Jideama, Noland et al. 1996; Sumandea, Pyle et al. 2003; Sumandea, Burkart et al. 2004; Sumandea, Vahebi et al. 2009)
Thr213	C	C	Raf-1	Unknown	(Pfleiderer, Sumandea et al. 2009)
Ser285	N	C	PKC	Reduce Myosin ATPase activity, myofilament force production and Ca2+ sensitivity	(Sumandea, Vahebi et al. 2009)
Ser285	N	C	ROCK-II	Reduce myofilament force development, Myosin ATPase activity and Ca2+ sensitivity	(Vahebi, Kobayashi et al. 2005)
Thr294	N	N	PKC	Reduce Myosin ATPase activity, myofilament force production and Ca2+ sensitivity	(Noland, Raynor et al. 1989; Jideama, Noland et al. 1996; Montgomery, Chandra et al. 2001; Sumandea, Vahebi et al. 2009)
Thr294	N	N	ROCK-II	Reduce myofilament force development, myosin ATPase activity and Ca2+ sensitivity	(Vahebi, Kobayashi et al. 2005)

Amino acid residues in cardiac TnT identified with phosphorylation regulations are summarized. The residue numbers are that in the sequence of human cardiac TnT with the first methionine counted. The phosphorylation of cardiac TnT at each of the residues is compared with its counterparts in fast TnT and slow TnT. C, conserved and N, non-conserved phosphorylatable residue in skeletal muscle TnT. Kinases responsible for each phosphorylation, potential functional effects, and references are also summarized.

Ser₂, a residue conserved at the N-terminus of all three TnT isoforms, is constitutively phosphorylated (Perry 1998). Little is known about the kinase and regulatory mechanisms for TnT phosphorylation at Ser₂ or its functional significance. When embryonic cardiac TnT is over-expressed in the adult heart of transgenic mice, Ser₂₅ encoded by the embryonic exon 5 is also constitutively phosphorylated (Zhang, Feng et al. 2011). Therefore, the N-terminal structure of TnT appears highly accessible by a highly active kinase at physiological conditions, and potentially resistant to phosphatases. The functional significance of N-terminal phosphorylation of TnT remains to be investigated.

Although protein kinase A (PKA) plays an important role in the phosphorylation regulation of multiple myofilament proteins, such as TnI, myosin binding protein C and titin, neither cardiac nor skeletal muscle isoforms of TnT is a physiological substrate of PKA. Nonetheless, in an in vitro study, cardiac TnT was demonstrated as a PKA anchoring protein, tethering PKA to the myofilament to facilitate phosphorylation of other myofilament components (Sumandea, Garcia-Cazarin et al. 2011).

Multiple Ser and Thr residues in the middle and C-terminal regions of cardiac TnT have been reported to be phosphorylated by protein kinase C (PKC), especially PKCα, PKCε and PKCξ (Katoh, Wise et al. 1983; Perry 1998; Dubois-Deruy, Belliard et al. 2015). PKC phosphorylation of cardiac TnT exerted a negative inotropic effect on decreasing the calcium-dependent actomyosin ATPase activity and myofilament force production, which was secondary to decreases in the binding affinity of TnT for tropomyosin-F-actin filament (Noland and Kuo 1992).

Mouse cardiac TnT was phosphorylated by PKC at Thr₁₉₇, Ser₂₀₁, Thr₂₀₆, Ser₂₀₈ and Thr₂₈₇, and phosphorylation of Thr₂₀₆ alone was sufficient to reduce myofilament calcium sensitivity and force production. In a study using reconstituted myofilaments, substitution of Thr₂₀₆ in cardiac TnT with Glu to mimic the negative charge of phosphorylation resulted in significant decreases of actomyosin Mg-ATPase activity, calcium sensitivity and myofilament cooperativity (Sumandea, Pyle et al. 2003). Phosphorylation of TnT at Ser₂₀₈ by PKCε enhanced cardiac contractility, which was reduced in heart failure due to decreased level of PKCε (Dubois-Deruy, Belliard et al. 2015).

In the conserved C-terminal region of cardiac TnT, phosphorylation sites were also identified for Apoptosis Signal-Regulating Kinase (ASK1) and Rho-Dependent Kinase 2 (ROCK2). Yeast-two-hybrid screen detected association of ASK1 and cardiac TnT (He, Liu et al. 2003). ASK1 was activated in cardiac muscle under stress conditions by proinflammatory mediators such as TNFα and ROS. Phosphorylation of cardiac TnT at Thr₁₉₄ and Ser₁₉₈, lead to an inhibition of the shortening and calcium transient in cardiomyocytes (He, Liu et al. 2003).

ROCK2 is a protein kinase associated with cardiac myofilaments. Mass spectrometry determined that ROCK2 phosphorylation of cardiac TnT at Ser₂₇₈ and Thr₂₈₇ (Vahebi, Kobayashi et al. 2005). Using skinned mouse ventricular muscle bundles, ROCK2 phosphorylation of myofilament proteins, especially cardiac TnT and cardiac TnI, reduced myosin ATPase activity and tension development (Vahebi, Kobayashi et al. 2005).

Together with the regulation of kinase activities, protein phosphatase 1 (PP1) (Jideama, Crawford et al. 2006) and protein phosphatase 2A (PP2A) (Dubois-Deruy, Belliard et al. 2015) dephosphorylate cardiac TnT at multiple sites and reverse the effects of phosphorylation on calcium sensitivity and myofilament force production.

5.2. O-linked GlcNAcylation

A recent study identified that Ser₁₉₀ of cardiac TnT is a substrate of O-N-acetylglucosamine modification (Dubois-Deruy, Belliard et al. 2015). O-GlcNAcylation of cardiac TnT was increased in a rat model of heart failure in correlation to a loss of cardiac muscle contractile function. Interestingly, O-GlcNAcylation of TnT at Ser₁₉₀ inhibits phosphorylation of TnT at Ser₂₀₈, suggesting a crosstalk between the two posttranslational modifications of TnT, possibly through intramolecullar allosteric competition for occupancy in the two closely positioned sites (Dubois-Deruy, Belliard et al. 2015).

5.3. Proteolytic modifications

Proteolytic regulation of TnT plays important roles in striated muscle cells in physiological as well as pathological conditions.

Maintaining normal stoichiometry of myofilament proteins

Troponin T undergoes rapid turnover with a half life of ~3.5 days in cardiac muscle (Martin 1981). When not incorporated in the myofilaments, TnT was rapidly degraded to avoid toxic effects (Jeong, Wang et al. 2009). The fast turnover and removal of surplus TnT protein in muscle cells is critical in maintaining protein stoichiometry of the contractile machinery (Feng, Hossain et al. 2009).

Caspase destruction

In apoptotic rat cardiomyocytes, cardiac TnT was cleaved by caspase 3 to generate a 25-kDa N-terminal truncated fragment, losing the N-terminal hypervariable region and a portion of the middle conserved region (Communal, Sumandea et al. 2002). In canine diaphragm muscle under hypoxia, fast skeletal muscle TnT was also cleaved by caspase 3, to generate a 28-kDa protein fragment (Simpson, van Eyk et al. 2000). The caspase 3-mediated cleavage of TnT was shown to attenuate myofilament force production by decreasing myosin ATPase activity.

Restrictive N-terminal truncation

The N-terminal variable segment of cardiac TnT can be cleaved by μ-calpain proteolysis during myocardial ischemia-reperfusion (Zhang, Biesiadecki et al. 2006). In contrast to the caspase 3 cleavage discussed above, this structure modification is restrictive and selectively deleting only the entire N-terminal variable region (amino acids1–71) and leave the conserved middle and C-terminal regions intact (Zhang, Biesiadecki et al. 2006). The N-terminal truncated cardiac TnT remains incorporated in the myofilaments and produces a functional impact (Zhang, Biesiadecki et al. 2006). The restrictive N-terminal truncation of cardiac TnT has been found in cardiac muscle in multiple mammalian species including mouse, rat and pig treated with acute ischemia-reperfusion (Zhang, Biesiadecki et al. 2006).

In transgenic mouse hearts expressing both cardiac TnT and fast skeletal TnT, N-terminal truncation can be induced by stress treatment for both TnT isoforms despite their differences in the amino acid sequence at the cleavage site (Zhang, Biesiadecki et al. 2006). Stress of ex vivo fatigue contractions resulted in N-terminal truncation of fast skeletal TnT in mouse diaphragm muscle with a higher level under higher duty cycles and in overloaded contractions (Feng et al. 2013). Therefore, N-terminal truncation of TnT may be an early response to myocyte injury when myofilament-associated calpain is activated by intracellular calcium overload.

Ex vivo working hearts of transgenic mice over-expressing N-terminal truncated cardiac TnT in the cardiac muscle exhibited moderately reduced left ventricular systolic velocity, which prolonged the time of left ventricular rapid ejection and increased stroke volume, especially at high afterload (Feng, Biesiadecki et al. 2008). Consistent with the modulatory effect on decreasing contractile velocity, biochemical studies demonstrated that similarly N-terminal truncated cardiac TnT altered TnT’s binding affinities for tropomyosin, TnI and TnC but preserved the overall calcium sensitivity and cooperativity of cardiac myofilament with moderately decreased maximum myosin ATPase activity (Pan, Gordon et al. 1991; Biesiadecki, Chong et al. 2007). Therefore, the N-terminal truncation of cardiac TnT by myofilament-associated μ-calpain is a novel regulatory mechanism for fine tuning troponin function in response to stress conditions. It provides an acute adaptation mechanism for the heart to compensate for the loss of systolic function against increased workload and/or in energetic crisis such as myocardial ischemia (Feng, Biesiadecki et al. 2008). Considering TnT’s half life of ~3.5 days in cardiomyocytes (Martin and Orlowski 1981), the N-terminal truncated cardiac TnT will be replaced by newly synthesized intact protein in a few days, allowing the regulatory effect to be reversible.

6. Mutations and abnormal expressions of TnT genes in myopathies

Cardiac and skeletal muscle myopathies have been linked to abnormalities in the TnT isoform genes. TNNT1 mutations result in nemaline myopathy (Jin et al., 2003), TNNT2 mutations are linked to hypertrophic (HCM) and dilated (DCM) cardiomyopathies (Thierfelder, Watkins et al. 1994), and TNNT3 mutations have been found in distal arthrogryposis (DA) (Zhao et al. 2011). Most of these myopathic mutations of TnT are in the conserved middle and C-terminal regions that contain binding sites for tropomyosin, TnI and TnC and presumably do not tolerate structural variations. Table 3 summarizes representative pathogenic mutations and aberrant splicing found in cardiac, slow and fast skeletal TnT.

Table 3.

Representative TnT gene abnormalities found in cardiac and skeletal myopathies

TnT abnormality	Phenotype	References
Cardiac TnT (TNNT2)
Ile79Asn	HCM	(Thierfelder, Watkins et al. 1994; Lin, Bobkova et al. 1996; Palm, Graboski et al. 2001)
Arg92Gln	HCM	(Thierfelder, Watkins et al. 1994; Marian, Zhao et al. 1997)
Arg92Leu	HCM	(Forissier, Carrier et al. 1996; Palm, Graboski et al. 2001)
Arg92Trp	HCM	(Moolman, Corfield et al. 1997; Fujino, Shimizu et al. 2001; Shimizu, Ino et al. 2003)
Arg94Leu	HCM	(Varnava, Baboonian et al. 1999; Palm, Graboski et al. 2001)
Arg94Cys	HCM	(D’Cruz, Baboonian et al. 2000)
Ala104Val	HCM	(Nakajima-Taniguchi, Matsui et al. 1997)
Arg130Cys	HCM	(Koga, Toshima et al. 1996)
ΔGlu160	HCM	(Harada, Takahashi-Yanaga et al. 2000)
Glu163Arg	HCM	(Koga, Toshima et al. 1996)
Glu163Lys	HCM	(Watkins, McKenna et al. 1995)
Ser179Phe	HCM	(Van Driest, Ackerman et al. 2002)
Glu244Asp	HCM	(Watkins, McKenna et al. 1995)
Exon 17 deletion (Δ14)	HCM	(Thierfelder, Watkins et al. 1994)
Exon 16–17 deletions (Δ28+7)	HCM	(Thierfelder, Watkins et al. 1994)
Phe110Ile	DCM	(Watkins, McKenna et al. 1995; Nakaura, Yanaga et al. 1999)
Arg141Trp	DCM	(Mogensen, Murphy et al. 2004; Mirza, Marston et al. 2005)
Arg141Trp	DCM	(Li, Czernuszewicz et al. 2001; Lu, Morimoto et al. 2003)
Arg205Leu	DCM	(Mogensen, Murphy et al. 2004)
ΔLys210	DCM	(Kamisago, Sharma et al. 2000; Hanson, Jakobs et al. 2002; Hershberger, Pinto et al. 2009)
Glu244Asp	DCM	(Nakaura, Yanaga et al. 1999)
Asp270Asn	DCM	(Robinson, Griffiths et al. 2007)
Lys273Glu	DCM	(Fujino, Shimizu et al. 2002)
Arg278Cys	DCM	(Watkins, McKenna et al. 1995; Morimoto, Nakaura et al. 1999)
ΔE96	RCM	(Peddy, Vricella et al. 2006; Pinto, Parvatiyar et al. 2008)
E136K	RCM	(Kaski, Syrris et al. 2008)
Slow skeletal muscle TnT (TNNT1)
ΔExon8	NM	(van der Pol, Leijenaar et al. 2014)
S108X	NM	(Marra, Engelstad et al. 2015)
Glu180X	NM	(Johnston, Kelley et al. 2000; Jin, Brotto et al. 2003)
L203X	NM	(Abdulhaq, Daana et al. 2015)
ΔExon14	NM	(van der Pol, Leijenaar et al. 2014)
Fast skeletal muscle TnT (TNNT3)
Arg63Cys	DA	(Zhao et al. 2011)
Arg63His	DA	(Dale et al. 2014)

Representative mutations in human cardiac TnT that cause hypertrophic (HCM), dilated (DCM) and restrictive (RCM) cardiomyopathies, and mutations in human slow and fast skeletal muscle TnT that cause nemaline myopathy (NM) and distal arthrogryposis (DA) are summarized.

6.1. TNNT1

Mutations in TNNT1 gene encoding slow skeletal muscle TnT have been identified in recently years with increasing clinical significance.

Point mutations

A nonsense mutation in the exon 11 of TNNT1 gene at codon E180 was found to cause a recessive form of nemaline myopathy with infantile lethality in the Old Order Amish in Lancaster, Pennsylvania, thus named the Amish Nemaline Myopathy (ANM) (Johnston, Kelley et al. 2000). Deletion of the C-terminal segment of slow skeletal muscle TnT by the E180X mutation causes a loss of the T2 region tropomyosin-binding site 2 (Figure 1). The truncated ssTnT is unable to form troponin complex and incapable of incorporating into the myofilaments (Jin, Brotto et al. 2003). The non-incorporated ssTnT fragment was effectively degraded in muscle cells (Wang, Huang et al. 2005) to avoid cytotoxic effect (Jeong and Jin 2009), which is consistent with the recessive phenotype of ANM patients.

Transgenic mouse models reproduced partial myopathic phenotypes of ANM (Feng, Wei et al. 2009; Wei, Lu et al. 2014). Animal model studies revealed that deficiency of ssTnT significantly decreased the contents of type I slow fibers in diaphragm and soleus muscles, accompanied by hypertrophic growth of type II fibers and increased muscle fatigability (Feng, Wei et al. 2009; Wei, Lu et al. 2014).

More recent case reports have identified another nonsense mutation in TNNT1 in a non-Amish population, which also causes severe nemaline myopathies. This nonsense mutation of codon S108 in exon 9 of TNNT1 gene reported in a Hispanic male patient in New York caused ANM-like recessive phenotypes, including severe respiration muscle weakness, type I fiber atrophy and compensatory hypertrophy of type II fibers (Marra, Engelstad et al. 2015). The similar phenotypes of the E180X and S108X mutations of TNNT1 in causing recessive nemaline myopathies based on their loss of the T2 region tropomyosin-binding site 2 (Figure 1) (Jin and Chong 2010) demonstrate the critical role of the two-site anchoring of troponin on the thin filament in the assembly and function of the thin filament regulatory system.

Aberrant splicing

Mutation in intron sequences may cause error splicing of TNNT1 pre-mRNA. A recent case report found in a Dutch nemaline myopathy patient that a combination of alleles with aberrant exclusion of exon 8- and exon 14-encoded segments in compound heterozygous mutations caused severe slow skeletal muscle atrophy and weakness (van der Pol, Leijenaar et al. 2014). The patient presented with phenotypes similar to that of Amish Nemaline Myopathy. The deletion of exon 8 segment partially destroys the T1 region tropomyosin-binding site 1 but preserved the high affinity binding site 2 (Jin and Chong 2010), and deletion of the exon 14-encoded C-terminal end segment would not directly affect either of the tropomyosin-binding sites (Figure 1). Therefore, more investigation is required to understand the pathogenic mechanisms of these aberrant splicing mutations of TNNT1.

A rearrangement in TNNT1 gene (c.574_577 delins TAGTGCTGT) was reported in 9 Palestinian patients from 7 unrelated families with recessively inherited nemaline myopathy (Abdulhaq, Daana et al. 2015). This mutation leads to aberrant splicing that causes a truncation of ssTnT polypeptide at L203. This mutation also presents as a recessive nemaline myopathy with phenotypes similar but not identical to that found in ANM. While ssTnT truncated at L203 would preserve both T1 and T2 region tropomyosin-binding sites (Figure 1), the truncated ssTnT will not be able to bind TnI and TnC (Figure 1). The recessive phenotype of L203 truncation demonstrates that the formation of troponin complex is essential for anchoring on the thin filament, a mechanism that preventing a potentially dominant toxicity of the truncated ssTnT to compete for tropomyosin-binding in the muscle cells of carriers. This hypothesis is worth investigating.

6.2. TNNT2

Numerous point mutations and aberrant N-terminal splicing of cardiac TnT have been identified as causes of cardiomyopathies. These abnormalities alter cardiac TnT’s interactions within the thin filament regulatory system, myofilament calcium sensitivity, and/or the myosin ATPase activity (Watkins, McKenna et al. 1995; Knollmann and Potter 2001; Harada and Potter 2004; Garcia-Castro, Coto et al. 2009; Lu et al., 2013) (Table 3).

Point mutations

Single amino acid substitutions Ile79Asn, Arg92Gln (Morimoto, Yanaga et al. 1998; Schwartz and Mercadier 2003), Arg92Leu, Arg94Leu (Palm, Graboski et al. 2001) located in the middle region of cardiac TnT were found to increase calcium sensitivity of cardiac myofilament force production and cause dominantly inherited hypertrophic cardiomyopathy in humans. Transgenic mouse cardiac muscle over-expressing cardiac TnT-I79N showed increased myofilament calcium sensitivity, diastolic malfunction, and increased susceptibility to arrhythmia (Baudenbacher, Schober et al. 2008).

Mutations Glu244Asp (Nakaura, Yanaga et al. 1999; Harada and Potter 2004) Lys273Glu (Fujino, Shimizu et al. 2002) and Arg278Cys (Theopistou, Anastasakis et al. 2004) in the C-terminal region of cardiac TnT increase calcium sensitivity and cause dominantly inherited hypertrophic cardiomyopathy.

Cardiac TnT mutations that caused dilated cardiomyopathy were also found in both middle and C-terminal regions of cardiac TnT. For example, single residue deletion of Lys₂₁₀ (Kamisago, Sharma et al. 2000) and Arg141Trp substitution (Li, Czernuszewicz et al. 2001) both lead to calcium desensitization of myofilament force production and cause dilated cardiomyopathy.

Cardiac TnT interacts with TnI through the I-T interface segment spanning Leu₂₂₄ to Val₂₇₄. Mutations E244D (Tobacman, Lin et al. 1999; Yanaga, Morimoto et al. 1999), K247R, D270N, N271I and K273E in the TnT-TnI interacting coiled-coil region, i.e., the I-T arm, disrupt the stability of troponin complex and lead to cardiomyopathies.

Aberrant splicing

Error splicing of cardiac TnT have been found in animal models to cause cardiomyopathies. For example, continuing expression of the embryonic exon 5 in adult cardiac TnT was found in dog, cat and guinea pig hearts in correlation with dilated cardiomyopathy (Biesiadecki, Elder et al. 2002). Although embryonic cardiac TnT is a normal variant of TnT instead of a mutation, chronic coexistence of embryonic and adult cardiac TnT in adult ventricular muscle generates desynchronized myofilament actions that decreases the energetic efficiency of the heart as a rhythmic pump, imposing pathogenic consequences (Feng, Chen et al. 2012).

Abnormal splice-out of the exon 8-encoded segment of turkey cardiac TnT and the counterpart, exon 7, in dog cardiac TnT are found to cause dilated cardiomyopathy (Biesiadecki and Jin 2002; Biesiadecki, Elder et al. 2002). The causal relationship was confirmed in transgenic mouse hearts over-expressing the equivalent cardiac TnT with exon 7-encoded segment deleted (Wei, Gao et al. 2010).

A cardiac TnI mutation R111C is found in wild turkey cardiac muscle in combination with the exon 8-deleted cardiac TnT (Biesiadecki et al. 2004). Transgenic mouse hearts expressing the equivalent mutation K118C or cardiac TnT with exon 7 deletion showed dominant negative effects on cardiac function (Wei, Gao et al. 2010). However, when cardiac TnI-118C was co-expressed with cardiac TnT exon 7 deletion as that occurs in the heart of wild turkeys, they mutually rescued each other’s dominantly negative phenotypes and restored cardiac function to near normality (Wei, Gao et al. 2010). This compound phenotype of mutations in the two subunits of cardiac troponin suggests that the structure-function relationships of TnT and TnI need to be investigated using integrative approaches.

It is worth noting that despite that an increasing number of TNNT2 mutations have been reported to date, no point mutation has be identified in the N-terminal variable region (Figure 4). This observation may indicate the hypervariable nature of the N-terminal segment of TnT, which would be tolerant to changes introduced by single amino acid substitutions. On the other hand, The N-terminal variable region functions as a regulator to modulate the overall molecular conformation and function of TnT, thus aberrant splicing of the N-terminal segment of cardiac TnT to introduce large structural changes is able to cause cardiomyopathies.

Figure 4. Pathological mutations of TnT.

Locations of single amino acid substitutions and deletions, as well as exon deletions and C-terminal truncations found in inherited human cardiac and skeletal muscle diseases are indicated on the linear maps of cardiac, slow and fast skeletal muscle TnT. The filled boxes indicate alternatively spliced exons, among which the developmentally regulated ones are in solid black. The pathological phenotypes some representative mutations are listed in Table 3. Majority of these mutations are located in the conserved middle region of TnT corresponding the to tropomyosin binding site 1. Despite the large number of pathogenic mutations reported to date, none has been reported in the N-terminal hypervariable region of TnT.

6.3. TNNT3

Fewer pathogenic mutations of fast skeletal muscle TnT gene (TNNT3) have been reported. A TNNT3 mutation (p.R63C) was found in a Chinese family with distal arthrogryposis (DA) phenotypes (Zhao et al., 2011). A recent exomal sequencing study identified another dominant TNNT3 mutation at the same residue (p.R63H) in 18 members of a large Indian family also with the phenotypes of distal arthrogryposis (Daly et al., 2014). The affected individuals exhibit significant variability in phenotypes, suggesting an interesting model to further understand the structure-function relationship of TnT and the penetration of pathogenic mutations.

The locations of these pathogenic mutations in the polypeptide chains of ssTnT, cTnT and fsTnT are summarized in Figure 4.

7. Summary and perspective remarks

Troponin T plays a central role in the calcium regulation of striated muscle contraction and relaxation. Three homologous genes have evolved in vertebrates to encode muscle type specific TnT isoforms. Alternative splicing and posttranslational modifications provide additional regulation of TnT structure and function during development and in muscle and heart adaptations to physiological and pathological conditions. Over half century of extensive research has provided a strong knowledge base for understanding the gene regulation and structure-function relationship of TnT. Mechanistic insights have been learned from TnT isoform expression, myopathic mutations, and splicing abnormalities, laying a foundation for translational research for the development of new treatment and prevention of cardiac and skeletal muscle diseases.

In the meantime, many important questions regarding the gene regulation and structure-function relationship of TnT remain to be answered. Some examples are: What is the mechanism for the feedback from muscle mechanic properties to the TnT gene regulation and protein modification? What is the functional significance of alternative splicing of specific exons of the TnT genes? What is the functional significance of the mutually exclusive splicing of fast TnT exons 16 and 17? And what is the precise position of the very C-terminus, middle, and especially the N-terminal variable region of TnT in the thin myofilament? Continued research work using advanced technologies will address these questions for ultimately understanding the evolution and regulation of TNNT isoform genes and the molecular mechanisms that govern the function of TnT in striated muscle contraction and relaxation.

Highlights.

• Troponin T (TnT) is a regulator of striated muscle contraction

• 3 homologous genes have evolved in vertebrates encoding muscle type TnT isoforms

• Alternative splicing and posttranslational modifications add variations of TnT

• TnT gene expression is regulated during development and adaptations

• This review summarizes the current knowledge and perspectives of TnT research

Abbreviations

ANM

Amish Nemaline Myopathy

ASK

Apoptosis Signal-Regulating Kinase

ATP

Adenosine triphosphate

CaMKII

Ca²⁺ /calmodulin-dependent protein kinase II

CMT

Charcot-Marie-Tooth disease

cTnT

cardiac troponin T

DCM

dilated cardiomyopathy

fsTnT

fast skeletal troponin T

HCM

hypertrophic cardiomyopathy

PKA

protein kinase A

PKC

protein kinase C

PP1

protein phosphatase 1

PP2A

protein phosphatase 2A

Raf-1

proto-oncogene, serine/threonine kinase

RCM

restricted cardiomyopathy

ROCK

Rho-Dependent Kinase

ROS

reactive oxygen species

ssTnT

slow skeletal troponin T

TnC

troponin C

TNFa

Tumor necrosis factor a

TnI

troponin I

TNNT1

human slow troponin T gene

TNNT2

human cardiac troponin T gene

TNNT3

human fast troponin T gene

TnT

troponin T