Use of animal models to understand titin physiology and pathology

Abstract In recent years, increasing attention has been paid to titin (TTN) and its mutations. Heterozygous TTN truncating variants (TTNtv) increase the risk of a cardiomyopathy. At the same time, TTNtv and few missense variants have been identified in patients with mainly recessive skeletal muscle diseases. The pathogenic mechanisms underlying titin‐related diseases are still partly unknown. Similarly, the titin mechanical and functional role in the muscle contraction are far from being exhaustively clarified. In the last few years, several animal models carrying variants in the titin gene have been developed and characterized to study the structural and mechanical properties of specific titin domains or to mimic patients' mutations. This review describes the main animal models so far characterized, including eight mice models and three fish models (Medaka and Zebrafish) and discusses the useful insights provided by a thorough characterization of the cell‐, tissue‐ and organism‐phenotypes in these models.


| INTRODUC TI ON
Titin is well-known as the largest sarcomeric protein expressed in skeletal and cardiac muscle. 1 It plays a crucial structural and functional role in sarcomeres. [2][3][4] In the last few years, an increasing number of inherited myopathies and cardiac disorders associated with titin (TTN) variants have been identified. 5 These disorders differ with a very large spectrum regarding inheritance (dominant or recessive), age at onset, progression and pattern of affected muscles. 5 Heterozygous carriers of titin-truncating variants (TTNtv) affecting exons constitutively expressed in heart have an increased risk of adult-onset dilated cardiomyopathy (DCM). [6][7][8] A secondary genetic or non-genetic hit is, however, usually required to develop the cardiac disease. 9 The pathomechanism underlying titin-related cardiomyopathies is still unclear: a reduced expression of wild-type protein and the stable expression of truncated titin protein have been recently reported analysing myocardial tissue samples from patients with titin-related DCM. 10,11 Biallelic mutations, mainly TTNtv with few non-truncating variants, cause skeletal muscle diseases with or without a cardiac involvement. 5,[12][13][14][15][16][17][18][19] The increasing number of patients diagnosed with a recessive titinopathies is facilitating the delineation of genotypephenotype correlations. 20,21 Finally, non-truncating variants in two specific exons (344 and 364) cause distinct, well-characterized, phenotypes (HMERF and TMD/LGMD2, respectively). [22][23][24] At the same time, spontaneous and genetically engineered animal models bearing variants in the titin gene have been characterized. This review aims to describe the main animal models so far studied, including eight mice models and three fish models (Medaka and Zebrafish) ( Table 1).

| TITIN: ROLE AND IMP ORTAN CE
Titin is giant protein that resides within the sarcomere in the striated muscle and heart. 1 It spans the entire I-and A-bands of the sarcomere, connecting the Z-disc end of the actin filament with the tip of the myosin filament, and running bound to the surface of the myosin thick filament, through the A-band to the M-line at the centre of the sarcomere. 2 The structural role of titin in the muscle fibres is well characterized ( Figure 1). Given its high number of interactions with other sarcomeric proteins, titin is a central hub that controls many of the mechanical properties of the sarcomere at rest and during contraction. 2,25 In addition, Titin is also important for sarcomere formation, mechanosensing and signal transduction. [25][26][27][28] The N-terminal portion of titin interacts with several structural and signalling proteins like nebulin, α-actinin, telethonin, actin binding proteins and filamin C. 29-31 I-band titin includes mainly repetitive immunoglobulin (Ig) domains and the so-called PEVK region, which is named after the proline (P), glutamate (E), valine (V) and lysine (K) rich content. This region can extend when mechanical force is applied, providing the extensible or "spring-like" function of titin. 1,4,32 A-band titin region is a non-extensible region that interacts with myosins. It is composed of repetitive Ig domains and fibronectin domains. The M-band contain the serine/threonine kinase domain, a central hub for many signalling ligands, and interacts with myomesin and obscurin originating a scaffold that link thick filaments at the Mline of the sarcomere. [33][34][35][36]

| T TN G ENE AND TR AN SCRIP TS
Human TTN gene includes 364 exons (363 coding and one additional non-coding exon). 1,37 Titin transcripts undergo alternative splicing that can theoretically produce more than 1 million isoforms. 1,15,38 In particular, the I-band region of Titin is prone to alternative splicing events, which regulate the inclusion or removal of Ig-domains, the length of the elastic PEVK element 32 and also the expression of two important signalling hubs, the N2A and N2B elements. 1,2,4 The presence of these two elements (N2A and N2B) is likewise used to classify titin isoforms in three main categories. In particular, the N2A isoforms (containing only the N2A element) are expressed in the skeletal muscles. The two mainly cardiac isoforms are classified by the presence of only N2B element (N2B isoforms), or both the elements (N2BA isoforms). 3,39 The N2A and N2BA isoforms typically include a higher number of Ig-domains and a longer PEVK than the N2B isoforms.
Moreover, three further isoforms (Novex-1, Novex-2, Novex-3) have been reported. Novex-1 and Novex-2 are similar to the N2B isoforms, except for the inclusion of an isoform-specific exon (exon 45 in Novex-1 and exon 46 in Novex-2). The Novex-3 isoform exclusively contains the N-terminal region of titin, because of an alternative stop codon in the exon 48. A recently discovered isoform, Cronos, lacks the N-terminal and it is expressed in foetal and adult cardiac tissue. 40,41 An inferred transcript (metatranscript) including all the coding exons (except the exon 48) is also commonly used

| MUSCUL AR DYS TROPHY WITH MYOS ITIS (MDM)
The muscular dystrophy with myositis mice (mdm) is by far the most studied titin animal model, widely used to characterize titin physiology in skeletal muscle. This spontaneous model contains a complex rearrangement (a deletion and a LINE-1 element integration within titin I-band region between the N2A and the proximal PEVK regions). The rearrangement leads to an aberrantly spliced transcript and, thereby, to a protein lacking 83 amino acids including the deletion of the N2A calpain-3 binding region. 45 Homozygous mice (mdm/ mdm) are smaller compared with their wild-type siblings and show kyphosis, rigid and limited gait, associated with a severe muscle degeneration and necrotic fibres with phagocytosis, that had been initially mis-interpreted as inflammation hallmarks (thereby, the name myositis). They develop an early onset progressive muscular dystrophy. Mechanical experiments have showed a significantly impaired muscle contractile function already at 6 weeks of age. 46 The mice die at 2 months of age, 45 due to the loss of diaphragmatic contractile function leading to respiratory failure.
The pathomechanism underlying the observed muscle phenotype has been delineated by several more recent studies. In 2016, Powers and colleagues suggested that, in psoas muscle, titin stiffness enhances the force by stabilizing the sarcomere during force development. This mechanism is lost in mdm mice in which the skipped domains in the N2A and in the proximal PEVK regions lead to a decrease in titin stiffness in an active sarcomere. 47 More recent studies on soleus confirmed that, during activation, titin is the only known sarcomeric non-cross bridge viscoelastic element that interacts at high affinity with Ca2+ and actin. 48,49 This interaction is mediated by the regions deleted in mdm mice. 48  Consequently, the mice heart shows the exercise intolerance and a left ventricle hypertrophy, mimicking the heart failure with preserved ejection fraction observed in some titin patients. 61,62

| FINmaj MICE
The human FINmaj mutation is a 11-bp deletion/insertion mutation located in the last exon of TTN. 23 The variant results in a 4-amino acid exchange in the C-terminal Ig domain M10 of the M-band titin.
FINmaj is a founder mutation in the Finnish population with a frequency close to 1/2000 individuals and causes the late onset distal myopathy named tibial muscular dystrophy (TMD). 23 Mice carrying the FINmaj mutation well recapitulate the phenotype observed in patients. 63 Heterozygous mice develop a late-onset mild progressive myopathy with dystrophic changes visible from 9 months of age in three muscles, tibialis anterior, biceps femoris and quadriceps. No significant functional impairment is, however, detected.
Homozygous mice show a more severe phenotype with an earlier onset (dystrophic features are visible in soleus already after 1 month of age). A dilated cardiomyopathy with heart muscle fibrosis and left ventricular dysfunction is only observed in the homozygous mice.
In patients as well as in the model mice, the mutation causes a pathological in cis cleavage of the last C-terminal domains of the mutated titin protein. A calpain-3 binding site is located in proximity in the cleaved off region and dysregulation of the proteolytic activity seems to play a crucial role in the pathology. 64

| ΔME X5 MI CE
ΔMex5 mice carry a homozygous deletion of Ttn second last exon (exon 363), an alternatively splicing exon encoding the insertion sequence 7 (is7). 65 Interestingly, the exon 363 usage is highly variable in anatomically different muscles. Skeletal muscles undergoing aerobic exercises usually show a higher expression of is7+ isoforms. 38,66 Similarly, heart main isoform expresses the 363 exon. 7 The c-terminal part of titin interacts with numerous partners and is7 is included in a binding site for calpain 3, an enzyme almost absent in the cardiac muscle. 67 The total absence of is7+ isoforms in the homozygous mice results in a dystrophic phenotype in those muscles where the exon is usually expressed (e.g. the soleus muscle). 65 The is7+ isoforms are highly expressed in heart, and the is7+ sequence, encoded by exon 363, is believed to be essential for proper cardiac function in mice, explaining the observed dilated cardiomyopathy in ΔMex5 mice. 68 Although the absence of is7 has a direct consequence only on the interaction with calpain-3, which is not expressed in heart, it is highly probable that M-line titin acts as a protein scaffold and, thereby, the absence of is7 may impact other interactions, partly explaining the observed pathology.

| FIS H MODEL S ( AN INNOVATIVE TOOL FOR THE INVE S TI G ATI ON OF TITIN M UTATI O N)
Zebrafish (Danio rerio) and Medaka (Oryzias latipes) are two fish models in which titin has a structure similar to the human one Table 2.
These animal models are, therefore, emerging as alternative cheaper models to study titin function and to characterize the effect of large-scale mutations on the cardiac and skeletal muscle phenotype.
Teleost fish underwent a whole genome duplication event which led, in zebrafish, to the formation of two copies of titin, ttna and ttnb.
In contrast, medaka has only one titin gene, which is thought to be composed of 219 exons and shows evolutionary highly conserved regions. 69 Because of the evolutionary distance between medaka and zebrafish (about 110 million years ago) and the consequent subfunctionalization of gene copies, the repertoire of the duplicated genes is different, and the two models are unlikely to show redundant phenotypes. Thus, the medaka fish model, due of its reduced genomic duplication and unique phenotypes attributable to specific mutations, is a promising model for future studies of titin mutations.
A recent review published by Celine F. Santiago et al. 70 has discussed in depth fifteen different zebrafish titin mutated models created to characterize the cardiac structure and function and to study genetic and molecular mechanisms in muscle disease (Table 2). This was possible thanks to new tools of imaging and "-omics" technologies for the cardiac phenotyping in small animal models. Conversely, to date just one medaka model has been reported with a titin mutation. Herein, we report two zebrafish models, which carry titin mutation linked to a cardiac (Pickwick m171 ) and a skeletal (runzel) myopathy, and the medaka cardiac model nsh.

| Pickwick m171
Pickwick m171 (pik m171 ) is a recessive lethal mutation discovered in zebrafish during a large-scale genetic screen. 71 The mutation found was a T > G transversion, located in the unique sequence of the cardiac N2B exon. The pik m171 heart develops normally during the first

| Runzel
Runzel (ruz) is a mutation in the N2A region, isolated after the nethyln-nitrosourea (ENU) screening, and causing a recessive muscle dystrophy. 72  New animal models carrying mutations resembling those found in patients would be useful to characterize the pathomechanisms underlying human diseases.
At the same time, skinned fibres from patients´ muscle biopsies could be used for mechanical experiments although technical aspects (e.g. sample collection, amount and quality of patients-derived material, the need for healthy sex-and aged-matched control muscles) may hamper a relevant outcome of these experiments.
Finally, 3D culture models from human cells are emerging as a potential alternative approach other than vertebrate model organisms. 73 3D cultures mimic the structure and the functionality of human muscle tissues. Although currently the physiological properties of the 3D cultures are not yet comparable with those found in vivo in tissues, they may soon become a very useful tool for muscle disease modelling. 74 Since each of these approaches presents issues that can compromise its effectiveness, their synergistic use could be optimal to understand how a specific titin mutation leads to the development of a given clinical phenotype.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.