**2. History of the discovery and study of titin/connectin by SDS gel electrophoresis technique**

Titin was discovered in 1979 by Kuan Wang and his coauthors [48] using gel electrophoresis. In macroporous polyacrylamide gel prepared according to Etlinger et al. [49] and containing 3.2% acrylamide, three new bands were found above the myosin heavy chain: a closely spaced doublet and a singlet band with faster mobility [48]. Using crosslinked myosin heavy chains (MHC, 205 kDa) as standards, the authors were able to estimate that each of these doublet bands (1 and 2) has a Mr. ∼ 1 × 106 . Proteins 1 and 2 appeared to be immunologically identical and were named titin 1 (T1) and titin 2 (T2). The third protein had Mr. ∼ 5 × 105 and was thereafter named nebulin [50].

Another group of investigators headed by Maruyama studied the properties of connectin – the protein they discovered [51]. The properties of connectin as a protein candidate for the elastic filaments in sarcomeres of striated muscles of vertebrate animals were intensively explored by this group of authors in the late 1970s [52–54]. In 1981, having conducted a comparative study of electrophoretic mobility, amino acid composition, and localization in myofibrils of titin and connectin, Maruyama and coauthors showed that the major high molecular weight component of connectin was identical with that of titin [55]. Using 1.8–3.0% polyacrylamide tube gels prepared according to Weber and Osborn [56], and crosslinked MHC as standards, the authors showed that the molecular weights of α-connectin (corresponding to intact molecules of titin-1) and β-connectin (corresponding to proteolytic fragments of T1–T2) of breast muscle of the chicken were 2.8 × 106 and 2.1 × 106 , respectively [57].

(**Figure 1**). Each half of myosin filament in sarcomere includes six titin molecules [14] with

**Figure 1.** Sarcomere structure of vertebrate striated muscles. (A) Scheme of sarcomeric structure. Thick filaments consisting mainly of myosin are located in the A-band. Thin filaments consisting mainly of actin are located in the I-band of the sarcomere and in the A-band, where they overlap with thick filaments. The H-zone is the central part of the A-band free from thin filaments. Titin molecules are arranged from the M-line to the Z-disc of the sarcomere. Transversal bands of myosin-binding proteins (H, C, X) in the A-band of the sarcomere are indicated. (B) Microphotograph of sarcomere of

Titin molecule consists of repeating immunoglobulin-like (IgC2) and fibronectin-like (FnIII) domains. Titin also contains a kinase domain in M-line, unique sequences N2A, N2B, and PEVK in I-zone and phosphorylation sites in Z-disc, M-band, and I-band of sarcomere [15].

The giant size of titin molecule and its location in all zones of sarcomere provide a basis for polyfunctionality of this protein. It has been shown that titin is a framework for the assembly of thick filaments and the sarcomere [16, 17]; is involved in maintenance of the highly ordered sarcomere structure [18, 19]; contributes to the passive tension developed by the muscle during stretching and develops the restoring force during sarcomere shortening [20–22]; is involved in the regulation of actin-myosin interaction [2, 4, 13, 23–31]. The results of recent studies suggest that the elastic protein titin, as a mechanosensor (strain sensor and stress sensor), plays a key role in intracellular signaling processes and in particular, participates in the regulation of muscle gene expression and protein turnover in sarcomere [2, 6, 32–37]. These

N- and C-ends overlapping in the Z- and M-line of the sarcomere, respectively [15].

demembranized rabbit lumbar muscle. Scale bar: 200 nm.

46 Electrophoresis - Life Sciences Practical Applications

Further electrophoretic studies of titin (connectin) using different types of gels (1.8% or 2.3–4% polyacrylamide tube gels, 2–12% gradient polyacrylamide slab gel) [58–63]) revealed differences in electrophoretic mobility of T1 (α-connectin) in cardiac and skeletal muscles of vertebrates animals (fishes, amphibians, reptiles, birds, mammals). In particular, plots of molecular mass versus mobility, assuming 2.8 and 2.4 MDa for T1 and T2 of the rabbit psoas, respectively, yielded the following set of values for T1: 2.8 MDa (adductor magnus), 2.88 MDa (longissimus dorsi, sartorius), 2.94 MDa (soleus, semitendinosus) [60]. Cardiac muscle displayed the smallest titin. Similar data were obtained by us using 2.5–9% gradient polyacrylamide slab gel (**Figure 2**). Based on data obtained the assumption on the existence of isoforms of T1 was made [60, 61].

Titin isoform analyses for 37 adult rabbit skeletal muscles showed sizes between 3.3 and 3.7 MDa [75]. N2BA titin isoforms in cardiac muscle of different mammals had sizes between

Peculiarities of SDS-PAGE of Titin/Connectin http://dx.doi.org/10.5772/intechopen.75902 49

Using 1% vertical agarose gel [70] at least four classes of cardiac N2BA titin isoforms were observed, of which two rat embryonic/neonatal forms (N2BA-N1, N2BA-N2) had sizes of 3710 and 3590 kDa. These isoforms were found during late embryonic and immediately post-natal period [76]. These were gradually replaced by adult forms (N2BA-A1, N2BA-A2) with sizes of 3390 and 3220 kDa, respectively [76]. Similar titin isoform transformations were observed in embryonic/neonatal hearts of rat and other mammals and reported by the researchers [74, 77–80].

Giant titin isoforms expressed in rat striated muscles with an RBM20 autosomal dominant mutation were reported [81–83]. The molecular masses of these isoforms were estimated from

Our group headed by Zoya Podlubnaya conducts a comparative study of titin isoform composition in mammalian striated muscles under conditions of hibernation, microgravity, and during the development of pathological processes [84]. Vertical agarose-strengthened 2.2% polyacrylamide gel prepared according to Tatsumi and Hattori [85] was used to separate titin

Our first experiments, conducted more than 10 years ago, showed that, in addition to N2A, N2BA, N2B and T2 bands, there exist one or two more high Mr. bands (named NT) [84, 86]. Staining the gels with ethidium bromide revealed no nucleic acids in the bands, although western blots with 9D10 antibodies revealed titin bands. The bands were visualized in the electropherograms of striated muscles of mammals, but in the electropherograms of striated muscles in other groups of vertebrates (amphibians and birds) revealed no NT bands [84].

The content of NT titins in muscles of animals and humans was as follows: Mongolian gerbil (8–14%), mouse (13–18%), rat (9–26%), rabbit (13–30%), ground squirrel (24–33%), and human (29–41%) [73, 84]. Using human and animal skeletal muscle myosin heavy chain (205 kDa) and nebulin (770–890 kDa), as well as the N2A titin isoform (∼3600 and 3700 kDa) of rabbit and human soleus as standards [60, 75, 87, 88], we estimated that the NT has a Mr. of ∼3.8–

67, 82, 83], but titin aggregates in gels could not be excluded either [69, 88]. Data published in 2003 demonstrated in electropherograms of the dog heart left ventricle, together with the known N2BA and N2B isoforms and T2-fragments of titin, the presence of higher molecular

We were also not absolutely sure that titin NT bands were not aggregates of its lower molecular weight isoforms and their fragments. If this were so, then the proteolytic cleavage of titin accompanied by an increase in the content of its fragments must result in the higher content of aggregates. Experiments on proteolytic cleavage of titin in muscle tissue under the influence

of endogenous proteases were performed to test this assumption [84] (**Figure 3**).

weight double protein bands that were named titin aggregates [70].

[73]. Expression of titin isoforms with these molecular weights is not excluded [66,

their electrophoretic mobility in 1% vertical agarose gel to be 3750 and 3830 kDa [83].

**2.2. New high molecular weight forms of titin in striated muscles of mammals:** 

3.25 and 3.4 MDa [72].

**aggregates or intact isoforms?**

isoforms and their fragments.

3.9 × 106

**Figure 2.** Molecular weights of T1 isoforms from rabbit striated muscles. Electrophoresis was performed in a gradient 2.5–9.0% polyacrylamide vertical gel (8 × 10 × 0.1 cm). (1) myocardium (left ventricle); (2) m. soleus; (3) logarithmic dependence of molecular weight on protein electrophoretic mobility in gel. T1 molecular weight was assessed by the following standards: Cardiac MyBP-C (150 kDa), as well as myosin heavy chains (MHC, 205 kDa), nebulin (770 kDa), and T2-fragment (2400 kDa) of rabbit skeletal muscles [60, 87, 88].

In 1995, the complete complementary DNA sequence of human cardiac titin was determined [64]. Further studies showed that the titin gene (TTN) consists of 363 coding exons, which can be differentially spliced and theoretically could generate more than one million splice variants in striated and smooth muscles of mammals [7, 65–68]. Adult striated muscles express three major titin isoforms: N2A in skeletal muscles (3.35–3.7 MDa), N2B, and N2BA in cardiac muscle (2.97–3.3 MDa, respectively) [65].

#### **2.1. Electrophoretic detection of titin isoforms**

To confirm that muscles contain N2A, N2B, and N2BA isoforms of titin, different macroporous gels (2–9.5% gradient polyacrylamide slab gel, 1% agarose slab gel, agarose-strengthened 2% polyacrylamide slab gel, horizontal 1.3% polyacrylamide gel strengthened with 0.5% agarose) were used [69–73]. It was shown that the T1 mobility varied greatly between skeletal and cardiac muscles from different mammals. The major T1 bands were ascribed to the titin isoforms N2B and N2BA in cardiac muscle and the titin isoform N2A in skeletal muscles. According to Western blot data with using antibodies against the N-terminal and the C-terminal ends of titin, it was revealed that the N2A, N2B, and N2BA bands represent full-length titin molecules (titin 1 – T1) [69, 74].

Titin isoform analyses for 37 adult rabbit skeletal muscles showed sizes between 3.3 and 3.7 MDa [75]. N2BA titin isoforms in cardiac muscle of different mammals had sizes between 3.25 and 3.4 MDa [72].

Using 1% vertical agarose gel [70] at least four classes of cardiac N2BA titin isoforms were observed, of which two rat embryonic/neonatal forms (N2BA-N1, N2BA-N2) had sizes of 3710 and 3590 kDa. These isoforms were found during late embryonic and immediately post-natal period [76]. These were gradually replaced by adult forms (N2BA-A1, N2BA-A2) with sizes of 3390 and 3220 kDa, respectively [76]. Similar titin isoform transformations were observed in embryonic/neonatal hearts of rat and other mammals and reported by the researchers [74, 77–80].

Giant titin isoforms expressed in rat striated muscles with an RBM20 autosomal dominant mutation were reported [81–83]. The molecular masses of these isoforms were estimated from their electrophoretic mobility in 1% vertical agarose gel to be 3750 and 3830 kDa [83].
