**3. Nebulin**

**Figure 3.** Schematic representation of the A-band region of titin, illustrating its motifs and cardiomyopathy-associated mutations. Mutations in the *TTN* gene within this region are shown relative to the domains in which they are found. Insertions or deletions predicted to cause frame shifts (fs) and single amino acid deletions (del) are noted in blue and yellow, respectively. A red background indicates nonsense mutations resulting in premature stop codons (ter). Splice site donor/acceptor mutations are shown in green. Missense mutations are shown with a magenta background.

**2.4. Cardiomyopathy linked mutations within the region of titin that extends into the M-**

Similar to the NH2-terminus of titin, the COOH-terminus remains relatively unaffected by cardiomyopathy causing mutations. A total of 6 mutations have been described within the portion of titin that extends into the M-band. Sequencing of DNA from patients with DCM and ARVC has identified 2 missense mutations localized to the M-band region of titin. Specifically, in two related individuals exhibiting late-onset DCM, an arginine to glutamine conversion at amino acid 32069 was identified (R32069Q) [21]. In addition, a patient diagnosed with ARVC possessed a methionine to threonine transition at codon 33291 (M33291T) [9]. These mutations localize to Ig146 and Ig152, respectively, however, their pathogenicity has not yet been determined. A recent study using population based analysis of DCM and HCM patients, identified 2 indels within the M-band portion of titin [5]. Phenotypic data regarding the subjects, as well as the mechanistic affects of the mutations are unavailable. Interestingly, 2 deletion mutations within the M-band region of titin were identified in 2 non-related families exhibiting early onset myopathy, affecting skeletal muscle, with fatal cardiomyopathy. Sequence analysis indicated a deletion mutation of 1 bp in exon 360 (Mex3) and an 8 bp deletion in exon 358 (Mex1) [25]. Both deletions left the titin kinase domain intact but resulted in premature stop codons at Ig domains 147 and 150 and a loss of the COOH-terminal 447 and

**band**

68 Cardiomyopathies

Nebulin is a giant (~500-800 kDa) sarcomeric protein of striated muscles [26]. Similar to titin, nebulin is oriented longitudinally across the sarcomere, spanning the length of the thin filament [27]. Its NH2-terminus extends to the pointed ends of thin filaments in the sarcomeric I-band, and its COOH-terminus resides within the Z-disc [28]. The nebulin gene, *NEB*, contains 183 exons and is the product of extensive gene duplication, resulting in a protein of highly repetitive domain structure [29]. Nebulin is mostly composed of tandem nebulin-repeats with the central motifs organized as super-repeats. In addition, nebulin contains a glutamine rich region at its NH2-terminus, as well as a serine rich region and a Src Homology 3 (SH3) domain at its COOH-terminus. The organization of the nebulin repeats complements the periodicity of actin filaments [28]. Consistent with this, alternative splicing of the *NEB* gene generates proteins of different sizes, which correspond to thin filaments of various lengths [30], [31]. In addition to its role in stabilizing thin filaments, nebulin has also been implicated as a regulator of thin filament length [32], [33].

nebulin-repeats that bind to F-actin and the tropomyosin-troponin complex, while A592E is located in the region that is incorporated into the Z-disc. Variants K60N and G202R found in nebulin-repeats 1 and 5, respectively, were identified in adult patients developing clinical manifestations of DCM. On the contrary variant Q128R, located in nebulin-repeat 3, was identified in a newborn patient diagnosed with DCM and endocardial fibroelastosis. The remaining variant, A592E, located in nebulin-repeat 16 was also found in a newborn displaying clinical features of DCM. *In vivo* studies using cardiac specific nebulette mutant mice were able to recapitulate human cardiac disease phenotypes and begun to unravel the mechanisms by which these mutations affect cardiac function [8]*.* Specifically*,* variants K60N and Q128R were embryonic lethal with hearts exhibiting structural abnormalities. Additionally, mutant mice carrying G202R or A592E variants resulted in left ventricular dilation and impaired cardiac function. These functional defects were coupled with improper localization of mutant nebulette resulting in dramatic structural alterations in I-band and Z-disc proteins. Taken together these studies suggest that nebulette is required for normal maintenance of the sarcomere and stability of the Z-disc and identifies *NEBL* as a contributor to the development

Cardiomyopathies: When the Goliaths of Heart Muscle Hurt

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

71

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

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

of ICD and DCM.

**4. Obscurin**

linkage to cardiomyopthies.

**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‐ sense mutations are shown with a magenta background.

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 [4], [8].

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

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 nebulin-repeats that bind to F-actin and the tropomyosin-troponin complex, while A592E is located in the region that is incorporated into the Z-disc. Variants K60N and G202R found in nebulin-repeats 1 and 5, respectively, were identified in adult patients developing clinical manifestations of DCM. On the contrary variant Q128R, located in nebulin-repeat 3, was identified in a newborn patient diagnosed with DCM and endocardial fibroelastosis. The remaining variant, A592E, located in nebulin-repeat 16 was also found in a newborn displaying clinical features of DCM. *In vivo* studies using cardiac specific nebulette mutant mice were able to recapitulate human cardiac disease phenotypes and begun to unravel the mechanisms by which these mutations affect cardiac function [8]*.* Specifically*,* variants K60N and Q128R were embryonic lethal with hearts exhibiting structural abnormalities. Additionally, mutant mice carrying G202R or A592E variants resulted in left ventricular dilation and impaired cardiac function. These functional defects were coupled with improper localization of mutant nebulette resulting in dramatic structural alterations in I-band and Z-disc proteins. Taken together these studies suggest that nebulette is required for normal maintenance of the sarcomere and stability of the Z-disc and identifies *NEBL* as a contributor to the development of ICD and DCM.
