**2.2. New high molecular weight forms of titin in striated muscles of mammals: aggregates or intact isoforms?**

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 isoforms and their fragments.

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].

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

**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),

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)

muscle (2.97–3.3 MDa, respectively) [65].

48 Electrophoresis - Life Sciences Practical Applications

[69, 74].

**2.1. Electrophoretic detection of titin isoforms**

and T2-fragment (2400 kDa) of rabbit skeletal muscles [60, 87, 88].

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– 3.9 × 106 [73]. Expression of titin isoforms with these molecular weights is not excluded [66, 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 weight double protein bands that were named titin aggregates [70].

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**).

whereas the N2A, N2BA, N2B, and T2 bands had sizes between 2100 and 2800 kDa [73, 84]. The last values corresponded to a set of values for T1 (α-connectin) and T2 (β-connectin) [57, 60, 88]. Similar data were obtained for vertical agarose-strengthened 1.9% polyacrylamide gels (**Figure 4**). The gel resolved a doublet NT band at 3300–3400 kDa for cardiac muscle and a singlet band of

Peculiarities of SDS-PAGE of Titin/Connectin http://dx.doi.org/10.5772/intechopen.75902 51

Results from western blots with Z1/Z2 antibodies against the N-terminal end and AB5 antibodies against the C-terminal end of titin revealed the NT bands were full-length titin molecules [89] (**Figure 5**). We, therefore, hypothesized that the NT bands are intact N2A, N2BA, N2B titin isoforms [89]. Although this requires further research, we cannot exclude the possibility that the NT bands are the other protein immunologically identical to titin, for example, a protein whose long thin filaments were revealed in the "shades" of rabbit psoas myofibrils remaining after sequential removal of myosin, actin, tropomyosin, troponins, and the minor M-band proteins [90] (**Figure 6**).

**Figure 4.** SDS-PAGE analysis of titin isoforms in striated muscles of ground squirrel (*Spermophilus undulatus*); a modified view from [89]. Vertical agarose-strengthened 1.9% polyacrylamide gel (14.5 × 16.0 × 0.15 cm) was used to separate the titin isoforms. (1) Myocardium (left ventricle); (2) m. soleus. T1 molecular weight was assessed by the following standards: MHC (205 kDa), nebulin (770–890 kDa), titin-2 (2100–2400 kDa) of rabbit and human striated muscles [60,

87, 88].

3600–3700 kDa for skeletal muscles of mammals.

**Figure 3.** Proteolytic changes in titin in ground squirrel muscles. Electrophoresis was performed in vertical agarosestrengthened 2.1% polyacrylamide gel (8 × 10 × 0.1 cm). (1) m. soleus (control); (2) m. soleus (proteolysis, 1 h); (3) left ventricle of heart (control); (4) left ventricle of heart (proteolysis, 30 min). Proteolytic cleavage of titin was performed under the influence of endogenous muscular proteases. To this end, small pieces of muscle tissue (20–30 mg) were held for 30–60 min at 25–30°C. Then, 2–3 mg pieces were taken from the muscle sample and placed into solubilizing solution (10 mM Tris–HCl, 1.2% SDS, 10% glycerol, 2% β-mercaptoethanol or 75 mM DTT, 8–10 μg/ml of leupeptin or E64, pH 7.0) for the extraction and further electrophoretic testing of the proteins. T3300 is probably the proteolytic fragment of NT titin with molecular weight of ~3300 kDa.

It was found that proteolysis of titin in m. soleus for 30–60 min resulted in a reduction (sixfold to sevenfold) of the content of NT titin and twofold reduction of the content of N2A titin (**Figure 3**, lanes 1 and 2). At the same time, a considerable increase in the content of T2 and appearance of a band with a molecular weight of ~3200–3300 kDa (T3300) were detected, which is probably a product of NT titin degradation.

Proteolysis of titin in cardiac muscle for 30–60 min resulted in a 2–3-fold decrease in the content of NT and N2BA (**Figure 3**, lanes 3 and 4). At the same time, the increase in the content of not only T2-fragments but also N2B isoform of titin was observed, which could be explained by the presence of fragments of NT and N2BA titins in this protein band. Densitometry data showed that the total titin content (relative to MHC content) in muscles as a result of 30–60 min proteolysis has not changed.

Thus, the results did not confirm our assumption that NT bands are aggregates of lower molecular weight titin isoforms and their fragments. However, these data did not exclude the aggregative origin of NT bands. Assuming that molecular masses of titin aggregates should considerably exceed 3800–3900 kDa, we decided to find out more about the differences in electrophoretic mobility of the observed bands. We developed a horizontal agarose-strengthened gel system using 1.3% polyacrylamide and 0.5% agarose [73].The gels showed that mobility of the NT bands, as well as other titin bands, varied greatly in different muscles.

Using human and animal skeletal muscle nebulin (770–890 kDa) as well as MHC (205 kDa) as standards the molecular masses of N2A, N2BA, N2B, T2, and NT titin bands were estimated. The results obtained were unexpected for us. The NT bands had sizes between 3230 and 3730 kDa, whereas the N2A, N2BA, N2B, and T2 bands had sizes between 2100 and 2800 kDa [73, 84]. The last values corresponded to a set of values for T1 (α-connectin) and T2 (β-connectin) [57, 60, 88]. Similar data were obtained for vertical agarose-strengthened 1.9% polyacrylamide gels (**Figure 4**). The gel resolved a doublet NT band at 3300–3400 kDa for cardiac muscle and a singlet band of 3600–3700 kDa for skeletal muscles of mammals.

Results from western blots with Z1/Z2 antibodies against the N-terminal end and AB5 antibodies against the C-terminal end of titin revealed the NT bands were full-length titin molecules [89] (**Figure 5**). We, therefore, hypothesized that the NT bands are intact N2A, N2BA, N2B titin isoforms [89]. Although this requires further research, we cannot exclude the possibility that the NT bands are the other protein immunologically identical to titin, for example, a protein whose long thin filaments were revealed in the "shades" of rabbit psoas myofibrils remaining after sequential removal of myosin, actin, tropomyosin, troponins, and the minor M-band proteins [90] (**Figure 6**).

It was found that proteolysis of titin in m. soleus for 30–60 min resulted in a reduction (sixfold to sevenfold) of the content of NT titin and twofold reduction of the content of N2A titin (**Figure 3**, lanes 1 and 2). At the same time, a considerable increase in the content of T2 and appearance of a band with a molecular weight of ~3200–3300 kDa (T3300) were detected,

**Figure 3.** Proteolytic changes in titin in ground squirrel muscles. Electrophoresis was performed in vertical agarosestrengthened 2.1% polyacrylamide gel (8 × 10 × 0.1 cm). (1) m. soleus (control); (2) m. soleus (proteolysis, 1 h); (3) left ventricle of heart (control); (4) left ventricle of heart (proteolysis, 30 min). Proteolytic cleavage of titin was performed under the influence of endogenous muscular proteases. To this end, small pieces of muscle tissue (20–30 mg) were held for 30–60 min at 25–30°C. Then, 2–3 mg pieces were taken from the muscle sample and placed into solubilizing solution (10 mM Tris–HCl, 1.2% SDS, 10% glycerol, 2% β-mercaptoethanol or 75 mM DTT, 8–10 μg/ml of leupeptin or E64, pH 7.0) for the extraction and further electrophoretic testing of the proteins. T3300 is probably the proteolytic fragment

Proteolysis of titin in cardiac muscle for 30–60 min resulted in a 2–3-fold decrease in the content of NT and N2BA (**Figure 3**, lanes 3 and 4). At the same time, the increase in the content of not only T2-fragments but also N2B isoform of titin was observed, which could be explained by the presence of fragments of NT and N2BA titins in this protein band. Densitometry data showed that the total titin content (relative to MHC content) in muscles as a result of

Thus, the results did not confirm our assumption that NT bands are aggregates of lower molecular weight titin isoforms and their fragments. However, these data did not exclude the aggregative origin of NT bands. Assuming that molecular masses of titin aggregates should considerably exceed 3800–3900 kDa, we decided to find out more about the differences in electrophoretic mobility of the observed bands. We developed a horizontal agarose-strengthened gel system using 1.3% polyacrylamide and 0.5% agarose [73].The gels showed that mobility of

Using human and animal skeletal muscle nebulin (770–890 kDa) as well as MHC (205 kDa) as standards the molecular masses of N2A, N2BA, N2B, T2, and NT titin bands were estimated. The results obtained were unexpected for us. The NT bands had sizes between 3230 and 3730 kDa,

the NT bands, as well as other titin bands, varied greatly in different muscles.

which is probably a product of NT titin degradation.

30–60 min proteolysis has not changed.

of NT titin with molecular weight of ~3300 kDa.

50 Electrophoresis - Life Sciences Practical Applications

**Figure 4.** SDS-PAGE analysis of titin isoforms in striated muscles of ground squirrel (*Spermophilus undulatus*); a modified view from [89]. Vertical agarose-strengthened 1.9% polyacrylamide gel (14.5 × 16.0 × 0.15 cm) was used to separate the titin isoforms. (1) Myocardium (left ventricle); (2) m. soleus. T1 molecular weight was assessed by the following standards: MHC (205 kDa), nebulin (770–890 kDa), titin-2 (2100–2400 kDa) of rabbit and human striated muscles [60, 87, 88].

(smears migrating near the bottom of the gel) than those at 60°C [70]. A temperature of 50–60°C for 10–20 min has been considered optimal for the extraction and preservation of intact titin [70, 88, 95]. Results of our studies demonstrated that heating of SDS sample at the said temperature may lead to artifacts in the content of titin. SDS samples of mammalian cardiac muscle heated at 60−65°C for 20 min had another N2BA/N2B ratio than those at 30–40°C [96]. We recom-

Peculiarities of SDS-PAGE of Titin/Connectin http://dx.doi.org/10.5772/intechopen.75902 53

It is suggested that titin has a tendency to aggregate during electrophoresis, especially in gel systems that use a stacking gel or discontinuous buffers [88]. Fritz et al. [93], as well as Greaser and Warren [97, 98], recommended the inclusion of β-mercaptoethanol in the top anodic buf-

There is some peculiarity that should be noted with regard to the preparation of agarosestrengthened 2% polyacrylamide gel. Tatsumi and Hattori [85] to prevent polyacrylamide polymerizing before agarose is polymerized, cooled the glass cell with agarose solution (40°C) for 5 min in ice water. Similarly, we added the agarose solution to glass cells that were pre-cooled to 8–10°C and left the gel for 10 min in the refrigerator at 5°C. Then, we kept the

It is recommended to perform electrophoresis using macroporous, agarose-strengthened polyacrylamide gels at low currents. Neagoe (of Wolfgang Linke's group) noted that the best separation of the high molecular weight proteins was obtained by running the electrophoresis overnight at 2 mA per 8.6 × 7.7 cm gel [99]. In our studies with the use of similar gels (8.0 × 10.0 × 0.10 cm), we perform electrophoresis at 3 mA for 40 min, then increasing the cur-

Granzier and Wang recommended to refresh the tank buffer once at 2.5 h to limit pH changes

In summary, it should be noted that SDS-PAGE of titin is quite difficult and not a routine procedure. Giant molecular mass and the susceptibility of titin to degrade during preparation significantly complicate the study of this protein by electrophoresis. It is necessary to know the three main rules for a successful study of titin by electrophoresis: (1) use protease inhibitors (leupeptin, E-64, protease inhibitor cocktail); (2) do not heat SDS samples higher than 40–60°C; and (3) judiciously select the type of the gel. Currently, the most suitable gels for analyzing titin are the following: (1) vertical agarose-strengthened 2% polyacrylamide gel [85]; (2) vertical 1% agarose gel [70]; and (3) horizontal agarose-strengthened 1.3% polyacrylamide gel [73]. It should be noted that we have obtained experimental evidence of existence in mammalian striated muscles of higher molecular weight isoforms of titin, named NT. According to our data, the development of pathological processes leads to the destruction of NT titins (**Figure 7**),

mended heating titin in SDS at 35–40°C for 30–40 min [73, 84].

**2.4. Other details of the electrophoretic study of titin**

gel for 30 min at 20°C and then for 2–2.5 h at 27°C.

caused by electrolysis during electrophoresis [88].

fer to prevent disulfide crosslinking.

rent strength up to 7–8 mA [96].

**3. Conclusion**

**Figure 5.** Western blotting of titin in striated muscles of ground squirrel; a modified view from [89]. As primary antibodies, the following were used: Z1/Z2 to N-end of titin molecule located in Z-disc of sarcomere (1, 2); AB5 to the part of titin molecule in A-disk located near the M-line of sarcomere (3,4); (1,4) m. soleus; (2,3) myocardium (left ventricle).
