Introduction

Dilated cardiomyopathy (DCM), the most common type of cardiomyopathies, is characterized by ventricular dilation, systolic dysfunction, and fibrosis [1,2,3]. DCM can be acquired (idiopathic or environmental) or genetically inherited via genetic mutations, which accounts for approximately 40–50% of DCM [1,2,3,4]. About 30 such gene mutations have been identified and the number of genes responsible for DCM continues to increase [1, 4, 5]. Some DCM is associated with prominent cardiac conduction system disease, often referred to as conduction disease associated with DCM [1, 6]. One of the major genes accounting for approximately 6–8% of DCM with cardiac conduction system disease is the lamin A/C (LMNA) gene [5,8,, 7–9]. DCM patients carrying LMNA mutations have been reported to have a worse clinical prognosis than DCM patients carrying different pathologic DCM-associated gene mutations [10, 11, 12•, 13]. Therefore, further investigations are needed to understand how LMNA mutations alter signaling pathways, which will then guide us to find more specific treatments for this life-threatening disease [14,15,16,17]. To better understand the underpinnings of genetic mutations leading to disease manifestations and the potential therapeutic implications, a comprehensive review of recent insights into the structure and function of LMNA genes is warranted. In this review, we discuss the pathophysiology and clinical spectrum of LMNA mutations leading to DCM and the potential therapeutic strategies based on the recent cellular and molecular understandings of this disorder.

Structure and genetic determinants of lamin proteins

Lamins are type V intermediate filament proteins and are known to serve as major structural components of the nucleus [18, 19]. There are two major types of lamins, A type and B type [18, 20, 21]. A-type lamins include lamins A and C, which are alternative splice variants arising from a single gene LMNA (the gene of our interest), while B-type lamins include lamins B1 and B2, products of two separate genes, LMNB1 and LMNB2 [22,23,24,25,26]. The human LMNA gene comprises of 12 exons on chromosome 1q21.2–21.3 that encode A-type lamins: A, AD10, C, and C2, via alternative splicing [21, 27]. Type A lamins are widely expressed in all differentiated somatic cells, and their various mutations are known to cause laminopathies [20, 22, 28, 29]. Lamin A (664 amino acids) and lamin C (572 amino acids) proteins are structurally composed of a globular N-terminus head domain, a central coiled-coil rod domain implicated in dimerization of the proteins, and a C-terminal tail domain that includes an immunoglobulin-like domain where various posttranslational modifications occur [20, 22]. Most lamins undergo various posttranslational modifications such as phosphorylation, glycosylation, prenylation, sumoylation, nethylation, and malonylation, all affecting various downstream signaling pathways [30]. Posttranslational modification is especially important to produce mature lamin A from prelamin A [20, 30, 31].

The lamins A and C are identical in their first 566 amino acids which are encoded by exons 1–9. Exon 1 codes for the amino terminal head domain as well as the first part of the central rod domain; the rest of the central rod domain is coded by exons 2–6. It is in the C-terminal tail domain where the difference arises between the two proteins [21,32,, 22, 28, 31–33]. The C-terminal tail domain of lamin A is encoded by a part of exon 10 followed by full exons 11 and 12, while the tail domain of lamin C has a full exon 10 but lacks exons 11 and 12 [21, 31]. Exon 12 in lamin A contains a CAAX motif (C = cysteine, A = aliphatic, X = any residue) which serves as a site for sequential posttranslational modification processing. The process starts with cysteine undergoing isoprenylation, followed by the cleavage of the AAX motif. The final step is the carboxy-methylation of the farnesylated cysteine, which is followed by the cleavage of the last 15 amino acids (including the farnesylation) by the zinc metalloprotease Ste24 homologue (ZMPSTE24) protease, producing mature lamin A [34,35,36,37,38,39,40,41,42,43,44]. ZMPSTE24 is a membrane-associated enzyme that localizes to both the ER membrane and the inner nuclear membrane [12•, 13, 34, 37, 38]. It can farnesylate substrates such as prelamin A for proteolytic cleavage [45, 46]. Farnesylation and methylation lead to hydrophobicity, which facilitates the localization of lamin A to the nuclear envelope [14]. Conceivably, alteration in any step of this process may lead to functional impairment of translation at the nuclei, partially due to the abnormal/mutated prelamin A accumulation. Disruption in these cleavage events has been associated with various diseases such as premature aging syndrome, including Hutchinson-Gilford progeria syndrome, restrictive dermopathy, and lipodystrophy, including mandibuloacral dysplasia (MAD-B) [43,48,, 47–49]. In contrast, lamin C lacks the 98 amino acids at the C-terminus that are present in prelamin A (thereby lacking the CAAX motif). However, it contains a unique 6 amino acid C-terminus encoded by a part of exon 10. Nevertheless, it contains various sites for the lamin binding proteins that affect various downstream signaling cascades [21,32,, 22, 28, 31–33].

Although the human LMNA gene was first identified in 1986, it was not until 1999 that the LMNA mutation was found accountable for Emery-Dreifuss muscular dystrophy (EDMD), suggesting its role in human disease [32, 50]. EDMD is a type of muscular dystrophy that is characterized by slowly progressive muscle wasting and weakness, early tendon contractures, and dilated cardiomyopathy with conduction system disease [51]. Since 1999, approximately 400 new gene LMNA mutations have been reported (http://www.umd.be/LMNA/) and associated with a wide spectrum of human diseases termed laminopathies [19, 28, 32]. Currently, laminopathies comprise four distinct groups, depending on the affected tissue: (1) striated muscles (dystrophy, heart), (2) adipose tissue (lipodystrophy), (3) nervous system, and (4) accelerated aging syndrome. LMNA mutations can manifest as a multisystem disorder or tissue-specific disorder affecting the four tissues listed above [28,53,54,55,, 52–56]. Most laminopathies arise from missense mutations and disruption in posttranslational modifications that influence the function of lamins. These functions range from structural support of the nucleus to various cellular processes including gene regulations, protein-protein interactions, and DNA repair [19, 20, 22, 57, 58]. They can be autosomal dominant, autosomal recessive, or X-linked [23, 28, 34].

Functional role of lamin proteins

Lamins are major architectural proteins located within the nuclear matrix and provide a platform for the binding of proteins and chromatin [33]. They are attached to the internal face of the inner nuclear membrane, thereby conferring mechanical stability [20, 21, 33, 59]. Beyond their significant mechanical supporting roles for nuclear structures, lamins are now known to contribute to DNA repair/replication, transcription, mediating cellular signaling, chromatin organization, and cytoskeletal interactions [47,61,62,63,, 60–64]. Expression of A-type lamins was also proposed to be developmentally regulated in a tissue-specific manner and to be implicated in terminal tissue differentiation [65, 66]. Therefore, new findings of distinct LMNA mutations, including laminopathies with tissue-specific dysfunctions and distinctive phenotypes, suggest that more than just defective nuclear stability and deformation may play a role in the development of laminopathies from LMNA mutations [16,68,69,, 50, 57, 58, 67–70]. Indeed, diverse conditions of laminopathies have been characterized with features including not only misshapen nuclei or nucleus instability but also disorganization of heterochromatin, DNA repair dysfunction, impaired proliferation or survival, disruption of cellular signaling pathway, cell migration, senescence, stress response, and improper interaction with cytoskeleton [23, 31, 32, 63, 71].

Nuclear envelope support

As elements of the components that make up the nuclear envelope, lamins were primarily studied for their mechanical supporting roles in cells and during mitosis [72, 73]. Studies have demonstrated that lamin A/C defects induce changes in nuclear morphology in a subset of cells, such as misshapen nuclei, nuclear pore clustering, mislocalization of associated proteins, and aberrant intranuclear lamin foci [59, 61, 74]. Without lamins, assembled cell nuclei are small and fragile. Compared with those from LMNA +/+ mice, fibroblasts from LMNA −/− mice have more malleable and fragile nuclei that are less resistant to physical compression in an isotropic manner [74,75,76]. Lamin mutants known to have muscular phenotypes also demonstrate deformable nuclei with impaired stability and decreased nuclear stiffness [74]. It has been shown that cells from LMNA mutant patients have a range of different nuclear morphological phenotypes, suggesting lamins have a role as structural proteins [28].

Organizing chromatin

Lamins can organize and regulate chromatin position within the nuclear envelope [77, 78]. Studies with cardiomyocytes and MEFs derived from LMNA −/− mice showed a partial loss of peripheral heterochromatin, ectopic chromosome condensation, and mispositioned centromeric heterochromatin, a phenomenon also observed in cells with mutant lamin A proteins [59,80,81,82,, 79–83]. There are at least two chromatin-binding regions in lamins: one is located in the tail region and the other is within the rod domain [84, 85]. It is thought that the interactions between chromatin and lamins are mediated through histones and/or chromatin-associated proteins [85, 86]. Lamins associate with scaffold/matrix attachment regions that are involved in transcription, DNA replication, chromosome condensation, and chromatin organization [87]. The genome regions that preferentially associate with lamins are termed lamin-A-associated domains (LADs) [88]. The significance of their interaction and the exact molecular mechanisms leading to disease have yet to be determined, but there is growing evidence of altered interaction between lamin and chromatin in various laminopathies.

Participating in DNA repair

For studying the role of lamins in DNA repair, cells from Hutchinson-Gilford progeria syndrome (HGPS) patients were found to have a delayed recruitment of the repair factor p53-binding protein (53BP1) to damaged DNA sites. These cells showed increased levels of the double-stranded break marker γ-H2AX and were more sensitive to DNA damaging agents [89]. LMNA mutant cells causing muscular dystrophy or progeria are also shown to alter DNA damage regulators such as ATR and ATM signaling pathways [90]. Furthermore, it is now believed that LMNA mutations primarily affect not only myofibers but also the efficiency of satellite cells in muscle repair and regeneration [91]. LMNA mutations may therefore increase nuclear fragility by disrupting the mechanical coupling between the cytoskeleton and the nucleus and, consequently, lead to a greater susceptibility to physical stress, especially in tissues exposed to mechanical strain such as skeletal and cardiac muscle [61, 70, 79].

Transcription regulator

More and more evidence indicates that nuclear lamins can modulate gene expression either by directly interacting with chromatin or by sequestering transcriptional regulators at the nuclear periphery [60, 92, 93]. Several experimental results indicated that lamins mediate transcriptional regulation [92, 94, 95]. Lamin expression coincides with RNA polymerase II activity that changes according to the stages of development, and alteration of nuclear lamin organization can inhibit RNA polymerase II-dependent transcription [94]. A-type lamin also associates with numerous other transcriptional regulators, such as Rb, Gcl, Mok2, cFos, and Srebp1, affecting gene expression by sequestration of these factors or by influencing the assembly of core transcriptional complexes [93, 95, 96].

Mediating nucleo-cytoskeletal connections

Lamins are considered an extended part of the LINC complex, which mechanically bridges nuclei with the cytoskeleton through lamin-interacting proteins that span the nuclear envelope [97,98,99,100]. The LINC complex is composed of two protein families—SUN (Sad1p/UNC-84) domain proteins at the inner nuclear membrane, where they in turn interact with a member of the nesprin family of proteins in the luminal space, and KASH domain proteins at the outer nuclear membrane [97, 100]. With two isoforms, Sun1 and Sun2, the SUN domains are conserved C-terminal protein regions about a few hundred amino acids long, followed by a transmembrane domain and a less conserved region of amino acids [101]. SUN proteins interact with the nuclear lamina, nuclear pore proteins, and other nuclear proteins at the nuclear interior and in the cytoplasm [102,103,104,105]. Nesprins span the outer nuclear membrane, where they associate with various cytoskeletal elements in the cytoplasm [105]. SUN domain proteins and nesprins together form the core of the LINC complex, which bridges the nucleus with the cytoskeleton to regulate proper function of transcription factors and gene expression [104, 105]. Any alteration in the LINC complex has been implicated for various nuclear functions including migration, positioning, morphology, and mechanics, and disturbed lamin function interferes with nuclear stability, gene regulation, and cytoskeletal functions [33, 61, 103]. Fibroblasts from mice with LMNA mutations linked to progeria demonstrated deformed nuclei and overaccumulation of protein Sun1, thereby negatively affecting lifespan and various tissues [106]. There is also evidence showing that proper expression and localization of nuclear lamin A/C and associated LINC complex are required for proper actin-cytoskeletal function during cell differentiation [107]. Studies also demonstrate that mutations in lamins A and C can disrupt LINC complex function and cause defects in skeletal and cardiac cells [108,109,110]. In addition to its role in muscle, proper nucleo-cytoskeletal coupling bridged by lamins is also essential during wound healing, inflammation, cell migration, cancer metastasis, and development [108,112,, 111–113]. Clinical outcomes and the molecular pathology in patients with laminopathies also indicated that the LINC complex was involved in LMNA mutations [76]. A-type lamin defects also affect different nuclear functions such as DNA replication, RNA transcription, and maturation by interacting with chromatin and many other transcription factors [98, 99].

Mediating cellular signaling pathway

Lamins also functionally interact with more than 30 direct and more than 100 indirect diverse proteins, indicating the function of lamins as a nuclear platform [33, 114, 115]. Moreover, the properties of variant proteins involved in these interactions may determine the tissue-specific roles of lamins [33, 116]. In addition, lamin A/C is also involved in a variety of signaling pathways affecting cell growth, survival, migration, and differentiation, including mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) pathways [60,118,, 117–119]. Through their interactions with the LINC complexes, cytoskeletal proteins, and chromatin, lamins regulate various signal transduction pathways that are important for diverse cellular functions [60, 120, 121]. Lamin and its associated nuclear proteins are shown to regulate the MAPK signaling pathway and its various downstream molecules as well as transforming growth factor-beta signaling pathways involving SMADs and other transcription factors [96, 122, 123].

LMNA mutations causing dilated cardiomyopathy

LMNA mutations are now known to account for 6–8% of dilated cardiomyopathy with conduction defect, portend a poor prognosis, and are associated with a high risk for sudden cardiac death [7,10,, 9–11, 12•, 13, 23, 124, 125]. Patients with LMNA mutations present with cardiac symptoms by mid-age (in their 20’s to 30’s) usually with mild low grade conduction system disease or atrial arrhythmia [7, 13, 126]. The conduction system disease gradually worsens and over 90% of patients develop dysrhythmia (bradyarrhythmias and tachyarrhythmias) with about half of patients needing either a pacemaker or ICD [7, 127]. There is a high incidence of sudden death (30%) and development of congestive heart failure (30%) [11, 13, 124, 125]. Moreover, almost half of the patients die suddenly before they reach the stage of overt heart failure [5, 11, 127]. A high cardiac disease penetrance and a high mortality were found in mutation carriers [7, 124, 125]. Male mutation carriers have a worse prognosis due to a higher prevalence of malignant ventricular arrhythmias and end-stage heart failure [125].

In addition to DCM and conduction disease disorder, LMNA mutations are also found in some patients with severe forms of arrhythmogenic right ventricular cardiomyopathy, a pathological pattern characterized by myocyte loss/fibro-fatty replacement, and reduction/absence of the intercalated discs of myocardium and LV noncompaction [128,129,130]. LMNA mutation carriers were also found to be associated with an increased risk of thromboembolic complications [131].

Cardiac effects of LMNA mutations have been demonstrated in a murine model as well [59, 79, 132, 133]. Targeted disruptions in mice led to development of cardiac and muscular dystrophy by interrupting nuclear envelope integrity [59, 79]. Homozygous (LMNA −/−) mice were also shown to exhibit significant growth retardation with premature death by 6–8 weeks of age with severe dilated cardiomyopathy with conduction disorder. They also demonstrated features similar to muscular dystrophy and suffered premature death by 6–8 weeks of age. These mice demonstrate nuclear deformation/instability as well as transport defects [79, 132]. LMNA knock-in mice carrying the H222P mutation, a missense mutation known to be responsible for EDMD in humans, developed dilated cardiomyopathy with conduction defects at adulthood in addition to muscular dystrophy. All of them died by 9 months of age with further histological analysis demonstrating extensive fibrosis and presumed altered gene expression from lamin mutation. Male mice had more prominent phenotypes and suffered earlier death compared to female mice [133].

Potential treatment options for LMNA cardiomyopathy

Clinical management

Currently, there is no specific treatment for laminopathies including LMNA cardiomyopathy. Current clinical management strategies for patients with LMNA cardiomyopathy are identical to those for patients with other cardiomyopathies or heart failure, which includes symptomatic and supportive treatment with pharmacologic and ventricular device therapies (neurohormonal antagonists, diuretics for congestion, vasodilators for hemodynamic unloading) [1, 3, 4]. Pacemakers can be considered in cases with the development of progressive conduction delays [1, 4, 5]. While sudden death from arrhythmias may be prevented by implantation of a defibrillator, progressive heart failure eventually becomes refractory to treatment, and heart transplantation is frequently necessary [1,2,3,4,5]. As patients with LMNA cardiomyopathy have shown to have a worse clinical course compared to non-LMNA cardiomyopathy, studies have tried to identify certain risk factors for sudden cardiac death among the LMNA mutation carriers for more aggressive therapy and earlier pacemaker/defibrillator placement [7, 124, 134]. Furthermore, in cardiomyopathy involving an additional system of laminopathies, medication for seizures and spasticity may be required for neuropathy, while physical therapy and/or corrective orthopedic surgery may be helpful for patients with muscular dystrophies [135,136,137].

Potential targeted pharmacologic therapies

Understanding how lamins control and alter signaling pathways holds great potential for therapeutic application in diverse laminopathies, including LMNA cardiomyopathy. Several mouse models have been used to study molecular pathways affected by LMNA mutations [138]. Identified signaling pathways deregulated by LMNA mutations include the MAPK pathway and mTOR pathway involving various downstream targets [118,144,145,, 139, 140•, 141, 142•, 143–146]. The MAPK/extracellular signal-regulated (ERK) pathway is activated by various stimuli that control signaling cascades that regulate cell proliferation, growth, differentiation, survival, migration, and apoptosis. It is expressed in all eukaryotic cells and disruptions in this pathway have been known to play a role in cancer and other numerous human diseases [117, 118, 147]. The MAPK pathway is also known to be involved in intracellular signaling of the ventricular myocytes. The MAPK pathway works as a multitiered pathway, involving various downstream target signaling molecules [148]. mTOR, the serine/threonine kinase, also plays an important role in regulating growth, proliferation, survival, and protein synthesis [149, 150]. It has been found that there is often cross-talk between mTOR and MAPK pathways [148].

LMNA knock-in mice carrying the H222P mutation, a missense mutation known to be responsible for Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy in humans, are known to develop dilated cardiomyopathy with conduction defects that lead to eventual death by 9 months of age [117, 118, 142•, 143]. In the hearts of these mice, hyperactivation of the MAPK signaling pathway including ERK, JNK, elk, and c-jun, which are all downstream components of MAPK cascades, was observed [118, 141, 142•, 143]. Treatment of these mice with various inhibitors of MAPK signaling pathways (inhibitors of MAPK/ERK, JNK, or both) demonstrated a delay in LV dilation and improvement of LV systolic function in mice with dilated cardiomyopathy [141, 142•, 143].

Moreover, it has been shown that proper interaction of A-type lamin with activated ERK1/2 regulates activation of junction protein connexin43 (Cx43). Without normal A-type lamins, Cx43 activation increases due to inappropriate phosphorylation of ERK1/2, resulting in decreased gap junction function that may decrease cell communication and contribute to the arrhythmic pathology associated with laminopathies [151, 152].

These results provide genetic evidence that ERK1 and ERK2 contribute to the development of cardiomyopathy in laminopathies [146]. Hyperactivation of the MAPK/ERK signaling pathway was observed in explanted human hearts with LMNA cardiomyopathy, indicating that these inhibitors hold a great therapeutic potential for human subjects with LMNA cardiomyopathy [153, 154]. Various inhibitors of the MAPK or mTOR pathway are already in therapeutic use to treat other pathological diseases such as cancer, chronic pain, and inflammatory diseases in humans [139, 148, 150]. Recently, a phase II trial commenced using ARRY-797, a selective oral inhibitor of the p38 MAPK, in patients with LMNA cardiomyopathy. The company’s phase I trial demonstrated a favorable outcome for patients on ARRY-797, showing improved cardiac function on an echocardiogram. The final outcome from this trial has yet to be determined, but this trial exhibits a viable therapy for LMNA cardiomyopathy by attenuating left ventricular dilatation and deterioration (www.ClinicalTrials.gov, Identifier NCT02057341).

LMNA mutant mice also exhibited hyperactivation of the mTOR pathway in affected tissues such as cardiac and skeletal muscle [133, 155, 156]. LMNA mutant mice treated with mTOR pathway inhibitors (rapamycin or temsirolimus) showed improvement in their LV size and cardiac function [155].

Potential cellular and molecular therapies

Identifying the precise molecular mechanisms of LMNA mutations leading to laminopathies affecting striated muscles is critical for developing new therapeutic strategies to prevent cardiac dysfunction and sudden death. Lamin mutations are known to alter functions of various transcription factors [92]. Cells in mice with lamin mutations linked to muscular dystrophy and DCM were shown to have altered function of transcription factor megakaryoblastic leukemia 1 (MKL1) due to abnormal nuclear-cytoskeleton dynamics. This was rescued by expression of one of the nuclear proteins, emerin [157]. In addition, expression of cardiac-specific lamin A transgene in LMNA −/− mice demonstrated improvement in cardiac function with a preservation of a functional conductive system [132]. These findings indicate a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in laminopathies and also implies a potential therapeutic strategy for laminopathies.

Insights from noncardiomyopathy LMNA mutations

LMNA mutations are also known to cause accelerating syndromes such as HPGS [53, 158]. The most common mutation is due to the deletion of the C-terminal region required for posttransitional modification [63, 159]. This leads to an increase in farnesylated lamin A which is shown to cause mitochondrial dysfunction, abnormal chromatic interactions, DNA damage, and cell instability in both in vitro and the mice model [34, 63, 159, 160]. Several studies have shown that farnesyl transferase inhibitors showed some attenuation of progeria-like symptoms as well as restoration of nuclear morphology [161,162,163,164]. Currently, lonafarnib (FIT inhibitor) is undergoing a clinical trial in patients with HGPS with promising data showing improvement not only in weight gain but also in cardiovascular stiffness, bone strength, and hearing (neuropathy) [165].

Conclusions

Lamin A/C mutations are frequently reported as a cause of cardiomyopathy, often causing sudden death at a young age before patients even reach clinically overt heart failure. Mutations of LMNA associated with laminopathies are only beginning to be understood. Studies have demonstrated an intricate complexity of lamin function and how it affects a diverse spectrum of cellular and molecular changes responsible for laminopathies including LMNA cardiomyopathy. Further investigations are needed to examine how alternations in the lamin structure regulate various cellular and molecular processing such as transportation of trans-factors, signaling pathways, and/or processes of posttranslation. Such examinations can facilitate rational understanding of the pathology of laminopathies in order to design therapeutic strategies.