**4. Obscurin**

**Figure 5.** Schematic representation of the nebulin superfamily members, illustrating their motifs and cardiomyopathy associated mutations. Mutations in the *NEBL* gene are shown relative to the domains in which they are found. Mis‐

In addition to being a member of the family of large sarcomeric proteins of striated muscle, nebulin is also a member of a family of actin-binding cytoskeletal proteins, which includes N-RAP, nebulette, LASP-1, and LASP-2 (Figure 5). The unifying domain of nebulin family members is the actin binding nebulin-repeat, of ~35 amino acids in length, each containing an SDxxYK motif [34]. For the remainder of the chapter, we will focus on nebulette as it is the only member of the nebulin family that has been linked to cardiomyopathies. Cardiac specific nebulette is functionally similar to nebulin whereby it aids in the stabilization of actin filaments [26]. Nebulette localizes to the Z-disc [35], where it interacts with the thin filament proteins troponin and tropomyosin [36]. Mutations in the nebulettte gene, *NEBL*, which cause disrup‐ tion of the stabilization of the Z-disc, have been linked to the development of cardiomyopathy

Nebulette's involvement in cardiomyopathies was first identified in the early 2000's when Arimura et al screened *NEBL* paired normal subjects and patients with idiopathic dilated cardiomyopathy (IDC) for mutations in the nebulette gene [4]. The study identified several polymorphisms in *NEBL* with one variant showing a high frequency in patients with nonfamilial IDC. Patients carrying this variant possess a missense mutation, N654K, in the 18th nebulin repeat of nebulette. The mechanism by which this mutation causes disruptions in the heart is unknown, however due to its location this mutation likely disrupts nebulette's incorporation into the Z-disc. Notably, this observation has brought about a new role for

Recently, more direct evidence for the involvement of nebulette in the development of heart disease was demonstrated in several patients diagnosed with DCM [8]. Linkage analysis revealed four sequence variations in the *NEBL* gene in regions encoding nebulin-repeats along the length of the molecule. Specifically, variants K60N, Q128R, and G202R are located in

nebulette as a genetic marker for patients with non-familial IDC.

sense mutations are shown with a magenta background.

[4], [8].

70 Cardiomyopathies

Obscurin is the third giant protein of the contractile apparatus of striated muscles. Similar to titin and nebulin the obscurin gene, *OBSCN,* gives rise to a large (~720 kDa; obscurin A) multidomain protein composed mainly of Ig and FN-III domains [7]. In addition, obscurin possesses several signaling motifs within its COOH-terminal half, including an IQ motif, an SH3 domain, as well as tandem Rho Guanine Nucleotide Exchange Factor (RhoGEF) and Pleckstrin Homology (PH) motifs. Similar to *TTN* and *NEB*, *OBSCN* is also subjected to alternative splicing giving rise to several isoforms of differing sizes (Figure 6) [37]. Specifically, the *OBSCN* gene gives rise to another large isoform, referred to as obscurin-B, which has a molecular mass of ~870 kDa. Obscurin B contains two serine/threonine kinase domains, which replace the non-modular COOH-terminus of obscurin A. The kinase domains may be ex‐ pressed independently as smaller isoforms, containing one or both kinase domains (sMLCK or tMLCK, respectively). Unlike its counterparts, obscurins surround the sarcomere at the level of the Z-disc and M-band, where they are appropriately positioned to interact with several ligands and participate in their assembly and integration into the sarcomere and internal membrane systems [7]. Despite its large size, it is only recently that we learn of *OBSCN*'s linkage to cardiomyopthies.

Although the role of *OBSCN* in cardiomyopthies is still unclear, several studies have docu‐ mented an upregulation of obscurins during cardiac hypertrophic responses to pressure overload and myopathic responses to mutations in titin [38]-[40]. In addition, up-regulation of different *OBSCN* gene products, including full length obscurin and several of the smaller MLCK variants was reported to occur in mice with myocardial hypertrophy induced by aortic constriction [38]. This increase in expression was mainly observed in obscurin isoforms that contained the RhoGEF and kinase signaling motifs, and occurred early in the hypertrophic response and also during hypertrophic growth. Concurrent with this, targeted loss of the obscurin RhoGEF domain resulted in myocytes lacking intercalated discs and in more severe cases in failure of the contractile filaments to organize into mature sarcomeres [41]. It is likely that upregulation of obscurins, is associated with the increase in contractile structures observed during hypertrophy, however, the mechanism by which this occurs remains unresolved.

titin, nebulette, and obscurin in cardiac muscle, and the effects of the identified mutations in their localization, activity, and regulation, it is not surprising that many human diseases of heart muscle have been linked to these proteins. Notably, a striking 50 missense mutations within *TTN, NEBL,* and *OBSCN* (Table 1) with an additional 16 splice site donor/acceptor mutations (Table 2) and 47 deletion or insertion mutations within *TTN* (Table 3) have been associated with the development of different forms of cardiomyopathy. The severity of these diseases can vary from moderate to severe, depending on the nature of the mutation. The characterization of these mutations and their effects on cardiac pathophysiology is just beginning to be elucidated, however it is clear that this is just the tip of the iceberg. Under‐ standing how these mutations alter sarcomeric structure and contractile activity could aid in improving clinical diagnosis and developing individualized therapies for cardiomyopathic

**Missense Mutations of Titin, Nebulette, and Obscurin**

Mutations of TTN DCM V54M\* Ig1 Z-disc Reduced binding to telethonin [18] HCM R740L\* Z-repeat 7 Z-disc Increase binding to α-actinin [19] DCM A743V\* Zq region Z-disc Reduce binding to α-actinin [18] DCM W976R\* Ig4 Z-disc Unknown [20] ARVC T2896I\* Ig16 I-band Unknown [9] HCM S3753Y# N2B I-band Increase binding to FLH2 [18] DCM Q4007X# N2B I-band Truncation of titin [18] DCM Q4249X^ N2B I-band Truncation of titin [6] DCM S4417N# Ig24 I-band Decrease binding to FHL2 [18] DCM G3470D\* PEVK I-band Unknown [23] ARVC Y8031C\* PEVK I-band Unknown [9] HCM R8500H\* PEVK I-band Increase binding to CARP [22] HCM R8604Q\* PEVK I-band Increase binding to CARP [22] ARVC H8848Y\* PEVK I-band Unknown [9] DCM C13771X^ Ig94 I-band Truncation of titin [6] DCM G16189X^ FNIII17 A-band Truncation of titin [6] DCM W16359X^ Ig100 A-band Truncation of titin [6] DCM R17295X^ FNIII25 A-band Truncation of titin [6] DCM R17470X^ Ig103 A-band Truncation of titin [6] DCM E17783X^ Ig104 A-band Truncation of titin [6] ARVC I16949T\* FNIII29 A-band Unknown [9] DCM C18789X^ FNIII36 A-band Truncation of titin [6]

**Region Effect Reference**

Cardiomyopathies: When the Goliaths of Heart Muscle Hurt

http://dx.doi.org/10.5772/55609

73

**Sarcomeric**

patients.

**Disease Mutation Region on**

**Protein**

More direct evidence for the involvement of *OBSCN* in the development of heart disease was demonstrated in a single patient with HCM [3]. Linkage analysis revealed a sequence variation in the *OBSCN* gene in the region encoding the site of interaction for the Z-disc region of titin (Ig58/59), specifically an R4344Q variant in the Ig58 domain of obscurin. *In vitro* studies showed that this variant resulted in decreased binding of obscurin to titin as well as mis-localization of obscurin to the Z-disc. Despite this single case, it suggests that, like titin and nebulette, mutations in the *OBSCN* gene lead to the development cardiomyopathies.

**Figure 6.** Schematic representation of the obscurin isoforms, illustrating their motifs and cardiomyopathy associated mutation. The missense mutation in the *OBSCN* gene is shown relative to the domain in which it is localized with a magenta background.
