The ONM provides the primary interface for the anchoring of the rod-like NE spectrin repeat proteins (nesprins). Nesprins are derived from the SYNE family of genes encoding four main proteins (nesprin-1, nesprin-2, nesprin-3 and nesprin-4), which can also undergo alternate initiation and termination to produce a number of isoforms that vary in structure and function. Full-length nesprins possess a C-terminal KASH (Klarsicht/ANC-1/SYNE-1 homology) transmembrane-spanning domain, required to provide anchorage within the nuclear bilipid layer (Zhang et al. 2001; Starr and Fischer 2005). Extending into the cytoplasmic domain is a series of spectrin repeats, the number of which varies depending on the nesprin isoform. The largest proteins are nesprin-1 and nesprin-2, encoded by SYNE1 and SYNE2, which extend deep into the cytoplasmic domain with 74 and 56 spectrin repeats, respectively. Both also have N-termini containing calponin homology (CH) domains that link to cytoskeletal actin filaments (Padmakumar et al. 2004; Zhen et al. 2002). Nesprin-3 and nesprin-4 are smaller protein isoforms with fewer spectrin repeats. They differ at the N-termini compared to nesprin-1 and nesprin-2 and link to intermediate filaments such as desmin, via plectin, and microtubules, respectively (Roux et al. 2009; Wilhelmsen et al. 2005). Nesprin isoform expression is also highly cell type specific. Some nesprin-1 and nesprin-2 isoforms show highest expression in muscle tissue types (Zhang et al. 2002) and are likely to play a major role in maintaining the function and integrity of striated muscle as evidenced by their involvement in Emery-Dreifuss muscular dystrophy and cardiomyopathy (Puckelwartz et al. 2010; Zhang et al. 2007).

The perinuclear space is occupied by the Sad1 and UNC (SUN) domain proteins, of which there are two main isoforms, SUN1 and SUN2 (Hodzic et al. 2004; Malone et al. 1999). SUN proteins form coiled-coil trimers, which are bound at their C-termini by peptides at the terminus of the nesprin KASH domain on the ONM interface of the NE. The SUN coiled-coil trimer extends across the luminal space of the NE penetrating through the INM and into the nucleoplasmic domain where the N-termini assume a globular shape and form a strong association with the nuclear lamins (Haque et al. 2006; Sosa et al. 2012).

The nuclear lamina is a meshwork of proteins lining the nuclear interface of the INM, consisting of type V IF proteins known as A- and B-type lamins, which form parallel coiled-coil dimers and head-to-tail dimers to produce a large network that covers the entire INM (Ben-Harush et al. 2009). The predominant A-type lamins are lamin A and lamin C which are alternate splice products of the same gene, LMNA (Lin and Worman 1993). Unlike lamin A, lamin C is translated as a mature protein, whereas lamin A is translated as a precursor, prelamin A, which must undergo a number of modifications before becoming mature lamin A (Davies et al. 2009). B-type lamins are lamin B1 arising from the LMNB2 gene and lamins B2 and B3 from the LMNB3 gene (Harborth et al. 2001). Whilst A-type lamins are thought to be specific to differentiated cell and tissue types, B-type lamins are thought to be present ubiquitously (Broers et al. 1997). Lamins have a postulated role in the regulation of gene expression, DNA damage repair, nuclear stability and structure and heterochromatin organisation (Misteli and Scaffidi 2005). To supplement these roles, the INM also has a set of distinct membrane-associated proteins that include lamina-associated polypeptide 2 (LAP 2), emerin and MAN1 (collectively termed the LEM domain proteins) (Lin et al. 2000). Alongside lamins, these proteins are also postulated to play roles in the regulation of gene expression and particularly in the case of emerin, in maintaining the integrity of the nuclear lamina (Bengtsson and Wilson 2004).

Emerin is the 254 amino acid product of the EMD gene and is a ubiquitously expressed integral protein of the INM (Bione et al. 1994; Manilal et al. 1996; Nagano et al. 1996). Structurally, emerin contains a 220 amino acid N-terminal nucleoplasmic domain, a single transmembrane domain and a short C-terminal tail situated in the NE lumen (Cartegni et al. 1997). As well as the aforementioned roles in gene expression and structural nuclear integrity, emerin is also hypothesised to have a role in regulation of cell cycle and nuclear assembly during mitosis and has a known association with β-catenin which is thought to restrict accumulation of β-catenin in the nucleus (Markiewicz et al. 2006).

The sequential tethering of these NE spanning proteins is thought to be crucial for functions including structural stability and nuclear form, nuclear positioning, chromatin scaffolding and facilitating rapid physically mediated communication to the nucleus, from the cytoplasmic and extracellular environment via mechanotransduction and mechanosignalling.

The general importance of the NE is highlighted by the unique set of pathogenic conditions that arise as a result of perturbation to components of the NE termed nuclear envelopathies. These include a subset of the laminopathies (Table 9.1), which are a set of tissue-specific disorders, often affecting striated muscle tissue and/or with cardiac involvement and displaying elements of premature ageing. Research into the characteristics and mechanisms of such diseases has helped identify a major role for the NE in cardiac cytoarchitecture and disease.

In the cardiomyocyte (CM), the LINC complex is postulated to mediate a physical interaction between the NE and the sarcomere. It is thought that within the cytoplasm this occurs via interactions between the actin-binding CH domain of nesprins, which can potentially bind actin of the sarcomere, but also via interactions between nesprins, plectin and desmin (Fig. 9.1). The importance of this link is twofold. Firstly, the nucleus is likely to be crucial to the structural integrity of the CM and therefore important in dealing with the perpetual mechanical stress imparted upon the cytoskeleton during contraction. Indeed, mutations in LINC complex components disproportionately affect tissues which are under mechanical strain suggesting that these links are crucial for the structural integrity of CMs. Secondly, the nucleus constantly senses the dynamic changes in the cytoplasmic (and potentially also the extracellular) domain due to mechanosignalling via the LINC complex and the cytoskeleton and consequently adjusts to such changes with appropriate gene expression responses (Meinke et al. 2014). Evidence suggests that these are the principal processes that become deregulated in disease settings, leading to the onset of cardiomyopathies.

Cardiac abnormalities associated with NE mutations are clinically varied and include dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC). Often these present in combination with other muscle phenotypes, such as limb girdle muscular dystrophy, LGMD, or more commonly Emery-Dreifuss muscular dystrophy, EDMD, reflecting the major influence of NE perturbations on mechanical tissues. EDMD is a condition in which patients experience early-onset contractures of the elbows, ankles and spine and endure subsequent humero-peroneal muscle wasting, and by adulthood, patients develop cardiomyopathy with conduction defects (Emery 1987, 1989, 2000). EDMD occurs by two primary means, both arising from components of the NE; one is described as X linked, in which mutations to the emerin gene EMD are identified as causative (Bione et al. 1994), and also autosomal dominant EDMD due to mutations in LMNA (Bonne et al. 1999). Of the NE components, LMNA mutations are currently thought to be the most common cause of NE-associated DCM. In fact, mutations in the LMNA gene are relatively commonly identified in patients with sporadic or inherited DCM and often lead to sudden cardiac death (van Berlo et al. 2005). As a result of this, LMNA is also the most rigorously studied NE protein in the context of heart disease.

HCM has also been described in conjunction with LMNA mutations. One instance involved a patient with familial partial lipodystrophy type 2 (FPLD2) caused by a missense mutation to LMNA that confers an amino acid substitution at residue 591 (Araújo-Vilar et al. 2008). This proband was also insulin resistant indicating a potentially wider role for lamins in heart disease, as perturbations to lamins may also be involved in diabetes and metabolic syndrome (Hegele 2000, 2003). Additionally, there was a case reported whereby a patient with the R644C mutation, arising in the C-terminal domain in close proximity to the prelamin A processing site, displayed pathogenicity consistent with HCM (Mercuri et al. 2005).

LMNA mutations have also been shown to be responsible for ARVC phenotypes. ARVC is characterised by pathological remodelling primarily of the right ventricle, in which myocyte death, inflammation and fibrofatty replacement of the myocardium are the main histological features (Sen-Chowdhry et al. 2010). ARVC arises primarily due to mutations in genes whose protein products contribute to the composition of the desmosomes such as desmoplakin, desmoglein, plakoglobin and plakophilin. Desmosomes are found at the plasma membrane in the intercalated disc (ID) regions where adjacent CMs join. The role of the desmosome is to tether the IFs of one cell to the cytoplasmic membrane of another to create a lattice structure that provides mechanical strength to tissue. However, a number of patients presenting with ARVC who did not have mutations to desmosomal genes were found to have LMNA mutations, suggesting that NE perturbations can influence CM cytoarchitecture at peripheral subcellular locations (Larsen et al. 2012; Quarta et al. 2011).

Mutations of the LMNA gene in humans have most frequently been shown to result in DCM leading to conduction defects resulting in arrhythmias and even leading to sudden cardiac death (Cowan et al. 2010). However, the recurrence of common DCM-causing mutations is low; the university mutation database states that there are currently 132 unique DCM-causing mutations to LMNA. As such, correlations between genotype and phenotype provide little idea on the mechanisms leading to cardiomyopathy. There are a subset of laminopathies which are caused by a few ‘hot spot’ mutations which lead to specific phenotypes and are likely to share mechanistic pathways (Scharner et al. 2010) including classical Hutchinson-Gilford progeria syndrome (HGPS), Werner syndrome, Charcot-Marie-Tooth disorder and MAD. However, the more common nuclear envelopathies, which are primarily striated muscle disorders, A-EDMD, LGMD1B and DCM, are caused by seemingly indiscriminate and promiscuous mutations which give little indication of the mechanistic milieu. For example, mutations that cause DCM have been found in all 12 exons of the LMNA gene. Moreover, mutations that cause DCM can also cause EDMD, LGMD1B and FPLD in separate individuals or can lead to any of these diseases with associated DCM. The level of pleiotropy seen with some of these mutations is unusual. For example, one curious mutation to the LMNA gene, R644C, displays extreme phenotypic diversity in the clinic (Rankin et al. 2008). These findings suggest that epigenetic or other extrinsic factors may be important in disease pathogenesis and progression. This diversity makes mechanistic analysis challenging, although some DCM-causing LMNA mutations have been investigated in order to provide some insight into the workings of LMNA-associated DCM and are discussed in a later section (Table 9.2).

Regarding nesprins, the situation is also complex. Mutations to SYNE1 can lead to autosomal recessive cerebellar ataxia due to nonsense mutations causing truncation of the protein (Li et al. 2014). However, SYNE1 and SYNE2 mutations have also been identified in probands with EDMD (Zhang et al. 2007) and DCM (Puckelwartz et al. 2010), and these missense mutations occur near the C-terminus of SYNE1/2 which encode muscle-specific isoforms that bind to other LINC complex components including emerin, lamin A/C and SUN (Zhang et al. 2007), suggesting that uncoupling of the LINC complex may contribute to cardiomyocyte dysfunction.

As mentioned above, mutations to emerin lead to EDMD with associated DCM. The mutations involved lead to loss of function of emerin and correlations with phenotype suggest that a fully functional emerin is required for skeletal muscle development and maintenance, whilst also important for cardiac muscle maintenance (Bione et al. 1994; Nagano et al. 1996; Patel et al. 2014).

Mutations to SUN proteins alone have recently been putatively associated to the onset of musculoskeletal and cardiomyopathic disease (Meinke et al. 2014). They have also been identified in combination with mutations to myosin-binding protein C, lamin A/C, emerin and LAP2α, in all instances leading to EDMD-like or cardiomyopathic phenotypes (Meinke et al. 2014; Li et al. 2014). This digenic pattern of mutation accumulation, whether sporadic or heritable, is believed to have disease-modifying effects, including early onset of disease and increased disease severity (Meinke et al. 2014). Mechanistically, it was shown, in vitro, that the identified mutations to SUN proteins led to defective myonuclear positioning via nuclear-microtubule uncoupling in human and C2C12 murine myotubes (Meinke et al. 2014).

There are two prominent hypotheses to explain why defects in the NE lead to cardiac cell dysfunction. The first of these suggests that mutations to NE proteins lead to structural changes that have a detrimental effect on the nucleus-cytoskeleton coupling and render cells more susceptible to mechanical stress (Worman and Courvalin 2004; Worman and Bonne 2007). The second suggests that mutations in NE proteins lead to abnormal gene expression (Worman and Courvalin 2004; Worman and Bonne 2007; Capell and Collins 2006). These hypotheses are not mutually exclusive and observations have identified the deregulation of multiple cell signalling pathways and cell maintenance systems, defects to gap junctions and fibrosis of cardiac tissue in a number of in vivo mouse models, suggesting that perturbations to the NE can have a disruptive influence on tissue organisation and integrity.