C in cardiomyocyte functions.

Biol. Cell (2014) 106, 346–358

Review

DOI: 10.1111/boc.201400033

Role of nuclear Lamin A/C in cardiomyocyte functions Monica Carmosino*†1 , Silvia Torretta†, Giuseppe Procino†, Andrea Gerbino†, Cinzia Forleo‡, Stefano Favale‡ and Maria Svelto†* *Department of Sciences, University of Basilicata, Potenza, Italy, †Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy, and ‡Cardiology Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy

Lamin A/C is a structural protein of the nuclear envelope (NE) and cardiac involvement in Lamin A/C mutations was one of the first phenotypes to be reported in humans, suggesting a crucial role of this protein in the cardiomyocytes function. Mutations in LMNA gene cause a class of pathologies generically named ‘Lamanopathies’ mainly involving heart and skeletal muscles. Moreover, the well-known disease called Hutchinson–Gilford Progeria Syndrome due to extensive mutations in LMNA gene, in addition to the systemic phenotype of premature aging, is characterised by the death of patients at around 13 typically for a heart attack or stroke, suggesting again the heart as the main site sensitive to Lamin A/C disfunction. Indeed, the identification of the roles of the Lamin A/C in cardiomyocytes function is a key area of exploration. One of the primary biological roles recently conferred to Lamin A/C is to affect contractile cells lineage determination and senescence. Then, in differentiated adult cardiomyocytes both the ‘structural’ and ‘gene expression hypothesis’ could explain the role of Lamin A in the function of cardiomyocytes. In fact, recent advances in the field propose that the structural weakness/stiffness of the NE, regulated by Lamin A/C amount in NE, can ‘consequently’ alter gene expression.

Lamin A structure and assembly in the nuclear envelope The nucleus is a dynamic organelle whose biochemical and functional properties are held within the nuclear envelope (NE), formed by the inner and outer nuclear membranes (INM and ONM), enclosing the perinuclear space (PNS). The INM and ONM are spanned by nuclear pore complexes (NPCs), large supramolecular structures that mediate bidirectional macromolecular trafficking between the cytoplasm 1 To

whom correspondence should be addressed (email [email protected]) Key words: Aging/senescence, Cardiomyocytes, Cellular differentiation, Nuclear membrane, Skeletal/cardiac muscle. Abbreviations used: AP-1, activator protein-1; BAF, barrier to autointegration factor; DCM, dilated cardiomyopathy; EDMD, Emery–Dreifuss muscular dystrophy; FHL2, four-and-a-half LIM protein-2; HGPS, Hutchinson–Gilford progeria syndrome; HP-1, heterochromatin protein 1; INM, inner nuclear membrane; KASH, Klarsicht, Anc-1 and Syne homology; LAP2, Lamin-associated polypeptide 2; LEM domain, LAP2, Emerin, MAN1 domain; LINC, linker of nucleoskeleton and cytoskeleton; LMNA, Lamin A/C encoding gene; MKL1, mechanosensitive transcription factor megakaryoblastic leukaemia 1; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1; NE, nuclear envelope; NPCs, nuclear pore complexes; ONM, outer nuclear membrane; PNS, perinuclear space; SRF, serum response factor; SUN, Sad1p and Unc-84 homology; TGF-β1, transforming growth factor beta-1; Tri-Me-K9H3, trimethylation of Lys9 on histone H3.

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and the nucleus. The endoplasmic reticulum (ER) together with INM and ONM forms a single continuous membrane system, in which PNS represents the extension of the ER lumen, and the contiguous ONM carries numerous bound ribosomes (Foisner, 2003; Gruenbaum et al., 2005). Under the nuclear membranes lies the nuclear Lamins, type V intermediate filaments, organised in thin elastic filamentous meshwork that lines the nucleoplasmic face of the INM, separating the NE from the nuclear matrix. The nuclear Lamins represent key structural components of the NE, maintaining the nuclear architecture and contributing to orchestrate important nuclear functions, such as gene regulation, DNA replication and chromatin organisation (LenzBohme et al., 1997; Sullivan et al., 1999; Liu et al., 2000; Dechat et al., 2008). Lamins, similar to other intermediate filament proteins, are formed by a tripartite structure consisting in a central α-helical rod domain flanked by nonα-helical N-terminal ‘head’ and globular C-terminal ‘tail’ domains. Two α-helical rod domain, through a characteristic heptad repeats of amino acids, wrap on each other forming coiled-coil homodimers,

Lamin A/C in cardiomyocytes physiology

which in turn can form ‘head-to-tail’ polymers that interact with each other forming antiparallel protofilaments. In addition, periodic and systematic distribution of charged residues promotes further lateral interactions, which then give rise to higher order structures, including 10 nm intermediate-like filaments (Stuurman et al., 1998). Another interesting feature of Lamins structure and assembly concerns the C-terminal globular domain, which is folded into a β-sandwich, similar to an immunoglobulin-like fold (Dhe-Paganon et al., 2002; Krimm et al., 2002). Lamins are tipically classified into A- and B-type, based on their structural features, isoelectric points, behaviour during cell division and patterns of expression in embryonic and adult tissues (Moir et al., 2000). A-type Lamins are widely expressed in the majority of differentiated somatic cells and are encoded by LMNA gene on chromosome 1q21.2–21.3. Alternative splicing events produce the two principal isoforms, Lamin A and Lamin C, indeed the LMNA gene product is widely indicated as Lamin A/C (Lin and Worman, 1993; Furukawa et al., 1994; Machiels et al., 1996). Lamin A and C entirely share the first 566 amino acids of their sequences, but differ for their C-termini. Lamin A, in fact, contains an extension of 98 amino acids, which encompasses a characteristic cysteine–aliphatic–aliphatic–any (CaaX) sequence. This motif is susceptible to complex series of post-translational modifications, including farnesylation and proteolytic cleavage necessary for the correct targetting to the NE (Holtz et al., 1989; Krohne et al., 1989; Kitten and Nigg, 1991). The newly synthesised Lamin C lacking of CaaX motif requires the presence of Lamin A for an efficient assembly into the nuclear lamina (Raharjo et al., 2001), giving to Lamin A the major physiological importance among the A-type Lamins. Lamin A/C also contains consensus sites for different kinases, whose phosphorylation directs Lamin A/C depolymerisation and disassembly during mitosis, allowing a proper nuclear membrane organisation in post-mitotic nuclei typical of muscle fibres (Heald and McKeon, 1990; Peter et al., 1990; Nigg, 1992). Lamins participate to a wide array of interactions that mediate their anchoring to the NE. The LAP2, Emerin, MAN1 (LEM) domain proteins represent the principal family of Lamin-binding proteins. The LEM domain consists in a 45-residue motif (Laguri

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Review et al., 2001) shared by several INM and intra-nuclear proteins, whose peculiarity consist in the ability to bind a conserved metazoan chromatin protein named barrier to autointegration factor (BAF). In vitro, it has been proven that BAF protein cross-links DNA molecules and binds histones (Margalit et al., 2007) and is involved in chromatin organisation, nuclear assembly and gene expression (Segura-Totten et al., 2002). The LEM domain proteins include emerin, MAN1, several LAP2 isoforms, LEM-2/NET25, and LEMs 3, 4 and 5 (Wagner and Krohne, 2007), among which emerin and LAP2 are the best characterised (Figure 1). Emerin is a ubiquitously expressed integral membrane protein of the INM (Manilal et al., 1996) that interacts with nuclear proteins regulating chromatin structure and transcription. Studies in vitro showed that emerin binds preferentially Lamin A/C (Lee et al., 2001) and requires it for proper NE targetting (Sullivan et al., 1999). Lamin-associated polypeptide 2 (LAP2) is a family of six alternatively spliced isoforms. LAP2α is located in the nuclear interior, where interacts with Lamins A/C (Dechat et al., 2000). LAP2α plays an important role in chromatin organisation by relocating throughout the nucleus during interphase (Vlcek et al., 1999) and to telomeric regions during mitosis (Dechat et al., 2004). Thus, a complex network of interactions between Lamin A/C, LEM proteins, BAF and, most likely, other INM proteins, is responsible for anchoring the chromatin to the NE and for organising chromatin higher order structure. The identification of this protein network involving Lamin A/C first suggested as primary function of this protein the regulation of gene expression within the nucleus. Another function of Lamin A/C, early identified, was related to NPCs spatial organisation in the NE (Aaronson and Blobel, 1975). Aaronson and Blobel isolated NPCs in association with Lamin A/C finding that pore complexes are interconnected and oriented by the Lamin A/C. By regulating import to and export from the nucleus, NPCs play a critical role in cell physiology enabling rapid yet selective bi-directional flow of material into and out of the nucleus to maintain cellular functions. As expected by the finding that Lamin A/C regulates the orientation and non-random distribution of the NPCs in the NE, the absence or mutation of Lamin A/C may profoundly alter NPCs

´ e´ Franc¸aise des Microscopies and Societ ´ e´ de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd 2014 Societ

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M. Carmosino and others

Figure 1 Schematic drawing of the Lamin A/C localisation in the nuclear envelope (NE) in cardiomyocytes showing Lamin A/C interacting proteins involved in both nuclear-cytoplasmic communication and in gene regulation.

localisation, function and selectivity (Dialynas et al., 2012).

Lamin A/C expression in contractile tissues B-type Lamins are ubiquitously expressed in all embryonic and adult cell types including embryonic stem cells (Harborth et al., 2001). Contrastingly, Lamin A/C is primarily found in differentiated cells. During mouse embryonic development, expression of Lamin A/C is first detected on day 9 in extraembryonic tissues and on day 12 in the embryo itself (Rober et al., 1989). Embryonic carcinoma cells generally express little or no Lamin A/C (Stewart and Burke, 1987; Prather et al., 1989). Lastly, certain adult cell types that are not fully differentiated also express little or no Lamin A/C (Lourim and Lin, 1989; Rober et al., 1989). The detection of Lamin A/C primarily in differentiated cells suggests that they might function to limit developmental plasticity by maintaining a cell’s differentiated phenotype. Lamin-B knockout mice die at birth as neurons apoptosis (Coffinier et al., 2010). Instead, Lamin-A/C knockout mice develop all tissues but die weeks after birth with growth retardation of connective tissues

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and also muscular dystrophy (Sullivan et al., 1999; Kubben et al., 2011; Jahn et al., 2012) that is very severe compared with the prototypical mouse model of muscular dystrophy (Dmdmdx), which lives for 2 years (Chamberlain et al., 2007). This observation suggests that Lamin A/C expression is essential for myocytes maturation and survival against the increasing in mechanical stress in adult muscles. In agreement, a recent and excellent study revealed that levels of the Lamin A/C in the NE scaled with tissue microelasticity showing high level of Lamin A/C in stiff tissues undergoing high physical stresses such as muscles and bone and very low level in soft tissue such as bone marrow and brain (Swift et al., 2013). In addition, the authors showed that stem cell differentiation into fat was enhanced by maintaining low Lamin A/C levels, whereas differentiation into bone was enhanced by high Lamin A/C level expression (Swift et al., 2013). In line with this observation, it has been reported that when human adipose tissue stem cells, in which Lamin A/C is completely absent, were exposed to cardiomyocytes cell extracts, they differentiated into beating cardiomyocytes showing as primary marker of such differentiation Lamin A/C expression (Gaustad et al., 2004).


These findings suggest that the expression of Lamin A/C is not involved in a general process of cell differentiation but specifically in lineage determination in favour of contractile and hard tissues. A real-time analysis of single-cell nuclear responses to stress in different cell types with different Lamin A/C levels demonstrated that while low Lamin A/C was insufficient to protect against extreme nuclear stresses that completely disrupted chromatin packing, the 30-fold higher Lamin A/C in stiff tissues, such as heart and skeletal muscles, would tend to impede rapid nuclear distension (Swift et al., 2013). Thus, high stresses and/or stress fluctuations typical occurring in a contractile tissue will not perturb the nucleus. The authors also shed light on the possible molecular mechanisms involved in the Lamin A/Cdependent cellular arrangement. They found that Lamin A/C expression causes accumulation of the mechanosensitive Yes-associated protein, a master transcriptional regulator (Zhao et al., 2010). An increase in Lamin A/C in stiff tissues also triggers the serum response factor (SRF) signalling pathway, whose gene targets control the actin cytoskeleton (Vartiainen et al., 2007) and structural genes involved in conferring the myogenic phenotype such as sarcomeric proteins (Balza and Misra, 2006). Moreover, Lamin A/C expression drives the translocation of the retinoic acid receptor into the nucleus to stimulate transcription of LMNA gene and the production of more Lamin A/C protein in the NE. A retinoic acidresponsive element within the Lamin A/C promoter has been identified in 2000 (Okumura et al., 2000) and the retinoic acid pathways have been already found to be involved in Lamin A/C expression in embryonic and differentiated cells (Olins et al., 2001). These recent findings clearly explain why mutations in LMNA gene cause a range of disease generically called laminoptahies, phenotipically affecting mainly skeletal muscles and heart, such as muscolar dystrophies and cardiomyopathies (Butin-Israeli et al., 2012). Before these recent findings, the answer to the question why Lamin A/C mutations affect contractile tissues and not all tissues was researched in tissue specific binding partners of Lamin A/C, whose homeostasis were affected upon Lamin A/C mutations. Actually, the study of interactome of Lamin A/C in different cell lines did not show differential Lamin

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Review A/C patterns of interaction in dependence of tissue types (Kubben et al., 2012). Rather, as demonstrated by Swift et al. only few months ago, the high and specific expression of Lamin A/C in skeletal muscles and heart makes the physiological role of Lamin A/C relevant in these tissues. Here, Lamin A/C acts as molecular mechanostat that relates to tissue stiffness and stress, clearly conferring protection against stress-driven rupture of nuclei and systematically affecting lineage determination.

The structural function of Lamin A/C in cardiomyocytes The structure and assembly of Lamin A/C expression in contractile tissues’ section, suggest that the primary role of Lamin A/C as structural support of the NE in contractile cells such as cardiomyocytes. In fact, the α-helical coiled coil structure assembled into rope-like fibres typically confers resistance to cells subjected to tension. The ‘mechanical stress’ hypothesis states that abnormalities in nuclear structure, resulting from mutations in Lamin A/C, weaken the nuclear lamina-envelope network and thus lead to increased susceptibility to cellular damage by physical stress. Lmna−/− mice develop dilated cardiomyopathy (DCM), and cardiomyocytes from these mice show abnormal nuclear architecture, which resulted multiglobulated suggesting an essential role of Lamin A/C in maintaining the correct nuclear shape (Nikolova et al., 2004). Moreover, these nuclei resulted less resistant to mechanic biaxial strain applied to the whole cells indicating that a markedly increased nuclear deformability and fragility in Lmna−/− cells (Nikolova et al., 2004). This has important implications for cardiomyocytes continuously undergoing to mechanical strain due to the contraction cycles. Impaired nuclear stability can lead to rupture of the nucleus, resulting directly in cell death. To determine whether apoptosis might be responsible for cardiac dysfunction in Lmna−/− mice, serial evaluation of DNA fragmentation in left ventricles tissue was performed in these mice. A significantly higher apoptotic index of Lmna−/− mice than wild-type and heterozygous Lmna−/+ mice was observed at 4–6 weeks of age but not earlier (Nikolova et al., 2004). This suggests that the cardiomyocytes apoptosis can be implicated at least in disease progression as


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observed in DCM and in cardiac conduction disorders in humans (James et al., 1996; Olivetti et al., 1997). In addition to the direct effect of nuclear rupture, altered nuclear mechanics can affect cells through impaired nuclear mechanotransduction. Lamin A/C interacts with INM proteins containing a SUN (Sad1p and Unc-84 homology) domain such as Sun1 and Sun2 (Starr and Han, 2003). Through the SUN domain, these proteins associate across the PNS with the KASH (Klarsicht, Anc-1 and Syne homology) domain of a protein family termed nesprins (Haque et al., 2006) located on the ONM; nesprins in turn connect to cytoskeletal components including actin, microtubules via dynein and intermediate filaments via plectin (Wilhelmsen et al., 2005), thus providing a link between the nucleoskeleton, the INM, the ONM, the sarcomere and the actin cytoskeleton (Figure 1). Lamin A/C with these interacting proteins forms the so-called LINC (linker of nucleoskeleton and cytoskeleton) complex, which is considered the molecular machinery involved in the nuclear–cytoplasmic communication and regulation of nuclear position (Haque et al., 2006). The main intermediate filament in cardiomyocytes is desmin (Tokuyasu et al., 1985) which links myofibrils radially along Z discs to the sarcomeres, longitudinally to the intercalated discs, and attach directly to the nuclear inner surface interacting with Lamin A/C (Figure 1). Interestingly, cardiomyocytes of Lmna−/− mice have marked disorganisation of the desmin filaments and increased PNS with regions in which the nucleus and adjacent cytoskeleton resulted completely disconnected (Nikolova et al., 2004). Moreover, Lmna−/− cardiomyocytes disproportionally increase in width when subjected to osmotic stress suggesting that altered nuclear anchoring compromises the scaffolding function of desmin (Nikolova et al., 2004). Intact physical and functional connections between the nucleus and cytoskeleton are required for effective mechanotransduction in cells. Indeed, these data support a model in which altered nuclear– cytoskeletal coupling alter the force transmission in cardiomyocytes with consequent impairment of contraction and the mechanic strain exerted onto the nucleus. Of note, the finding of desmin disorganisation in cardiac biopsy tissue from an individual in whom

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DCM and conduction-system disease was caused by a missense Lamin A/C mutation (Sebillon et al., 2003) is consistent with the hypothesis that these changes in nuclear–cytoskeletal coupling may actually be pathogenic. Moreover, the expression of the Lamin A/C N195K mutant variant, causing DCM in transgenic mice, results in the disruption of intercalated disc and sarcomeric and gap junctions organisation in mice cardiomyocytes (Mounkes et al., 2005). At an intercalated disc, the cell membranes of two adjacent cardiomyocytes are extensively intertwined and bound together by gap junctions and desmosomes. These connections help stabilise the positions of the cells relative to each other, the maintaining of the 3D structural integrity of the tissue and the electrical signal cell-to-cell propagation. Thus, the intercalated discs ensure proper function of the heart with efficient ejection of blood with each contraction. Intercalated disk distruption observed upon Lamin A/C mutant expression, indeed, may be also responsible of the conduction defects typically associated with DMC observed in both mutant Lamin A/C transgenic models and Lamin A/C mutant carriers (London, 2001). Additionally, a second Lamin-related protein network involving nesprins may regulate the nuclear– cytoskeletal coupling. Loss of Lamin A/C binding to nesprin could exacerbate desmin-induced changes in cytoskeletal tension. Recently, mutations in LMNA gene causing DMC showed different degree of Lamin A/C–Nesprin-2 interaction and causing LINC complex alterations (Yang et al., 2013), reinforcing the hypothesis that the disruption of the Lamin A/C interaction with one or more elements of the LINC complex may affect the cytoskeleton-nucleus mechanotransduction. Interestingly, cardiomyocytes seems to be more vulnerable than skeletal muscle cells to Lamin A/C haploinsufficiency in both patients and mice models. Indeed, genotype–phenotype correlations using LMNA Universal mutation database (http://www.umd.be/LMNA/) showed that 67 % of nonsense and truncating mutations, which are putatively dominant thus leading to lower lamin A/C levels, cause cardiac diseases without muscle involvement. The different sensitivity to Lamin A/C haploinsufficiency between the heart and skeletal muscles might due to structural and functional differences


of these two tissues. The heart contracts in a ‘twist’ way, generating torsion, whereas skeletal muscles generate unidirectional shortening. In addition, distinct organisations of the cellular cytoarchitecture and nuclei positioning (central in cardiomyocytes versus subsarcolemmal in muscle) result in singular force applied on cardiac nuclei compared with myofibres. Indeed, this may explain, in addition, the higher susceptibility of cardiac muscle to nuclear mechanical defects and deformations induced by a loss of Lamin A/C function.

Lamin A-associated signalling pathways in heart tissue The ‘gene expression’ hypothesis on the laminopathies pathogenesis proposes that defects in NE proteins lead to tissue-specific pathogenic changes in gene expression (Hutchison, 2002). The interplay of Lamin A/C and associated proteins with signal transduction pathways constitute an important mechanism by which the NE regulates gene expression in cardiomyocytes. Lamin A/C is involved in a wide spectrum of intermolecular interactions that affect signal transduction pathways. To date, numerous studies implicate NE proteins as regulators of the activity and/or availability of components of the MAPK (Muchir et al., 2007a), Akt/mTOR (Choi et al., 2012) signalling cascades and SRF (Ho et al., 2013) in cardiomyocytes. Signalling via these pathways has been identified to be defective in several laminopathies contributing to the understanding of the role of Lamin A/C in gene regulation in physiopatological conditions. The pathogenesis of Emery–Dreifuss muscular dystrophy (EDMD) and DCM, both due to LMNA gene mutations, is associated with perturbed MAPK signalling, as abnormal activation of ERK, JNK and p38α was observed before the onset of significant cardiac impairment in the hearts of LmnaH222P/H222P knock-in mice, a model of autosomal EDMD that features DCM (Muchir et al., 2007a; Muchir et al., 2012). Several downstream target genes (Elk1, Bcl-2, JunD, Elk4, c-Jun) are activated by MAPKs in hearts from these mice (Muchir et al., 2007b). Activation of these targets might consequently regulate expression of additional genes, including those encoding proteins involved in sarcomere structure, cardiomyofibre organisation, and other aspects of heart function

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Review (Gillespie-Brown et al., 1995; Thorburn et al., 1995). Hyperactivation of ERK1/2 also occurs in the hearts of emerin-deficient mice, a model of x-linked EDMD with DCM (Muchir et al., 2007a). The role of ERK1/2 hyperactivation in the pathogenesis of DCM is further supported by the finding that the cardiac impairment in LmnaH222P/H222P mice is blocked by treatment with MAPK signalling inhibitors before the appearance of clinical symptoms (Muchir et al., 2009; Wu et al., 2011). In addition, germline deletion of Erk1 from mice with the cardiomyophaty-causing LmnaH222P/H222P mutation leads to a transient improvement of heart function at 16 weeks of age. However, the improved cardiac function was abrogated at 20 weeks of age concurrent with an increased activity of Erk2 (Wu et al., 2014). More work is needed to clarify the mechanisms by which expression of mutant Lamin A/C and emerin deficiency lead to ERK1/2 hyperactivation. Some findings suggest that ERK1/2 interacts with Lamin A/C at the nuclear periphery and participate in the rapid induction of transcription factors such as activator protein 1 (AP-1) (Gonzalez et al., 2008), known to be involved in key cellular process such as cell proliferation, death, survival and differentiation (Shaulian and Karin, 2002). Therefore, alterations in Lamin A/C expression might perturb NE structure sufficiently to directly affect ERK1/2 activity and thus its several downstream signalling targets. One of the specific and important features of LmnaH222P/H222P mice is the extensive fibrosis observed in both cardiac and skeletal muscles, a feature that was also present in patients with striated muscle laminopathies (Arimura et al., 2005). A significant body of literature indicates that the transforming growth factor beta-1 (TGF-β1) is a powerful initiator for fibrosis, extracellular matrix deposition and alteration of gene expression in heart (Rosenkranz, 2004). The intracellular effectors of TGF-β1 signalling, the Smad proteins, when activated via TGF-β receptors, translocate into the nucleus where they regulate gene transcription. Briefly, receptor-activated Smad proteins, such as Smad2 and Smad3, are phosphorylated and form a heteromeric complex with Smad4. These phosphorylated Smad2/3–Smad4 dimers then translocate to the nucleus, mediating the target gene responses (Rosenkranz, 2004).


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Interestingly, hearts of LmnaH222P/H222P mice showed abnormal intranuclear accumulation of phosphorylated Smad2/3 proteins in cardiomyocytes, which may explain the extensive heart fibrosis (Arimura et al., 2005). This intranuclear accumulation of Smad proteins most probably is due to the abnormal nuclear shuttling of Smad proteins through NPCs whose selectivity can be compromised by the mutated Lamin A/C. Interestingly, it has been reported that Lmna−/− mice and Lamin A/C N195K mutant expressing cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1) (Ho et al., 2013), a myocardin family member that is pivotal in cardiac development and function (Olson and Nordheim, 2010). Normally, MKL1 is localised in the cytoplasm by binding to cytoplasmic G-actin and constitutive nuclear export. Mitogenic or mechanical stimulation triggers RhoA-mediated actin polymerisation, liberating MKL1 from G-actin and exposing a nuclear localisation sequence within the actin-binding domain of MKL1 (Pawlowski et al., 2010). Increased nuclear import, coupled with decreased export, causes accumulation of MKL1 in the nucleus, where it coactivates SRF. SRF, then, turns on genes regulating cellular motility and contractility, including vinculin, actin and SRF itself (Vartiainen et al., 2007) and structural genes involved in conferring the myogenic phenotype such as sarcomeric proteins (Balza and Misra, 2006). Importantly, cardiac sections from Lmna−/− and LmnaN195K/N195K knock-in mice had significantly reduced fractions of cardiomyocytes with nuclear MKL1 (Ho et al., 2013), confirming MKL1 translocation defects in vivo and implicating altered MKL1 signalling in the development of cardiomyopathies in these animals. Cardiac tissues from Lmna−/− mice had also lower SRF and actin transcript levels than those of wildtype littermates, and activation of SRF expression in response to left ventricular pressure-overload was impaired in Lmna+/− mice, demonstrating disturbed MKL1-SRF (Ho et al., 2013) mechanosignaling in vivo. As consequence of the impaired mechanosignaling, Lamin A/C deficient or defective cardiomyocytes undergo apoptosis faster than normal cardiomy-

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ocytes under mechanical stress (Lammerding et al., 2004). The expression of DCM-causing Lamin A/C E82K mutation in mouse heart tissues activates the two major signalling pathways of apoptosis. In particular, expression of this mutant variant increased the expression of FAS, accompanied with the activation of caspase-8/caspase-3 and caspase-9-dependent release of cytochrome c from mitochondria to cytosol (Lu et al., 2010). Interestingly, the apoptotic fate seems to involve earlier the conduction system cardiomyocytes (Wolf et al., 2008). Mechanical stress was unlikely to be significantly different in myocytes that populate or surround the cardiac electrophysiologic system. However, a different susceptibility to apoptosis might reflect intrinsic molecular properties that distinguish conduction system myocytes from atrial and ventricular myocytes. Because Lamin A/C sequesters cFos at the NE of fibroblasts and suppresses the DNA binding activity of transcription AP-1 by affecting cFos and cJun dimerisation (Ivorra et al., 2006), reduced levels of Lamin A/C protein could have consequences on downstream transcription. Since it is possible that atrio-ventricular nodal myocytes, like other excitable neural cells, are particularly sensitive to AP-1 mediated transcriptional regulation, including caspase activation and stress-induced apoptosis (Herdegen and Waetzig, 2001), mutations that reduce Lamin A/C levels could activate pro-cell death signals selectively in these specialised myocytes.

Lamin A/C and cardiomyocytes senescence Mutations in the LMNA gene are responsible for the premature aging disease Hutchinson–Gilford progeria syndrome (HGPS) (De Sandre-Giovannoli et al., 2003). The most prevalent HGPS mutation (heterozygous Gly608 →Gly608 with C changed to T) leads to a splicing defect and consequent generation of a truncated, dominant loss-of-function Lamin A/C isoform (De Sandre-Giovannoli et al., 2003). HGPS patient cells have various defects in nuclear structure and function (Goldman et al., 2004). They are characterised by dysmorphic nuclear shape, increased DNA damage and down-regulation of several nuclear proteins, including the heterochromatin protein


HP1 and the LAP2 group of Lamin A/C-associated proteins (Goldman et al., 2004). Furthermore, HGPS cells have altered histone modification patterns, including reduced heterochromatin-specific trimethylation of Lys9 on histone H3 (Tri-Me-K9H3) (Scaffidi and Misteli, 2005). After the discovery of progeria syndrome, it was not clear how HGPS related to normal aging and whether Lamin A/C played any role in the physiological aging process. Interestingly, in 2006, Scaffidi and Misteli determined whether HGPS-like nuclear defects occurred in cells from normally aged individuals. Multiple skin fibroblast cell lines from old (81–96 years) individuals consistently showed nuclear aberrations similar to those seen in HGPS cells. Whereas most cells from young individuals (3–11 years) showed robust staining for HP1, LAP2s and Tri-Me-K9H3, a significant subpopulation of nuclei in cells from old individuals had reduced signals, similar to previous observations in HGPS cells (Scaffidi and Misteli, 2006). Successively, age-related changes in Lamin A/C expression ware analysed specifically in mice cardiomyocytes (Afilalo et al., 2007). The expression of Lamin A/C in cardiomyocytes of old (24 months) versus young (4 months) C57Bl/6J mice, using a well-validated mouse model of aging and Lamin B1 as a control. Immunohistochemical and immunofluorescence analyses showed about seven-fold reduced expression of Lamin A/C in cardiomyocyte nuclei of old mice. Moreover, Lamin A/C distribution was scattered peripherally and perinuclear in old mice, whereas it was homogeneous throughout the nuclei in young mice. Western blot analyses confirmed about 50% reduced expression of Lamin A/C in nuclear extracts of old mice. Echocardiographic studies showed increased left ventricular wall thickness with preserved cavity size (concentric remodelling), increased left ventricular mass and a slight reduction in fractional shortening in old mice (Afilalo et al., 2007). These latter data add an important dimension to this study by showing that the morphological and functional correlation of the histopathological findings is consistent with the expected changes of myocardial aging. Accordingly, in both Lmna−/− -deficient (Nikolova et al., 2004) and Lmna+/− -insufficient (Wolf et al., 2008) mouse models, animals were born with normal functioning hearts, but eventually developed premature cardiac aging with various degree of atrioventric-

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Review ular block, atrial arrhythmias, DCM, and ventricular tachycardia, closely resembling to those observed in humans with heterozygous LMNA mutations and in old mice cardiomyocytes. Interestingly, the hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway has been observed in Lmna−/− (Ramos et al., 2012) and LmnaH222P/H222P knock-in mice (Choi et al., 2012) contributing to the heartand muscle-specific defects in these mice. Moreover, the inhibition of this pathway in both mice models can significantly counteracts this dysfunction and ultimately improves mice survival (Choi et al., 2012; Ramos et al., 2012). The first indication that the mammalian target of rapamycin (mTOR) pathway modulates mammalian ageing came from longevity studies as part of the National Institute on Aging’s Interventions Testing Program. Rapamycin was found to significantly extend lifespan in a genetically heterogeneous strain background at three independent test locations (Harrison et al., 2009). The mTOR pathway positively regulates cell growth and proliferation by promoting many anabolic processes while limiting catabolic processes such as autophagy, a physiological process in which the cell degrades damaged or excess cellular components through the lysosomal machinery. Activation of the mTOR pathway can be a beneficial response in muscle and cardiac tissue when hypertrophy is needed for instance. In cardiac tissue, the hypertrophic response is activated by hemodynamic overload and provides temporary relief for heart function. In Lmna−/− mice, however, the activation of mTORC1 did not induce any cardiomyocyets hypertrophic response (Ramos et al., 2012). Most likely this latter effect is due to the disorganisation of the desmin filament network and accumulation of desmin aggregates in these mice (Nikolova et al., 2004). Desmin, a major cytoskeletal protein in cardiac and muscle tissue, which connects the contractile apparatus to other organelles of the cell (including the mitochondria, lysosome and nucleus), is necessary for the cytoskeletal rearrangements required for adaptive hypertrophy (Hein et al., 2000). However, the hyperactivation of mTORC pathway in the absence of Lamin A/C is accompanied with the inhibition of the autophagy contributing to the disease phenotypes observed in Lmna−/− mice (Ramos et al., 2012). Many studies have shown that autophagy activation


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can prolong the lifespan of organisms from yeast to flies (Simonsen et al., 2008) and that autophagy activation is important for lifespan extension (Eisenberg et al., 2009; Morselli et al., 2010). Autophagy maintains cellular homeostasis by clearing damaged or toxic proteins/organelles and promotes cell survival under periods of starvation or increased energy demand by recycling its own cellular components. Indeed, in Lamin A/C lacking cardiomyocytes, such as senescent cardiomyocytes as well as unfunctional Lamin A/C-expressing cardiomyocytes, the inhibition of autophagy due to mTOR hyperactivation can be responsible for energy deficit or accumulation of toxic proteins/organelles in cardiomyocyets inducing senescence and ultimately cellular death.

Concluding remarks Here, we highlighted different biological functions of Lamin A/C with a specific look at the cardiomyocyte functions. Several models and experimental approaches are used for analysing structural and gene-regulatory functions of Lamin A/C in heart: Lamin A/C silencing, Lmna−/− mice, Lamin A/C mutants knock-in mice, morphological analysis of heart biopsy from Lamin A/C mutants carriers. Based on these studies, two working hypotheses have been proposed to explain the physiological role of Lamin A/C in cardiomyocytes. The ‘structural’ hypothesis proposes that Lamin A/C reinforces NE, which, in contractile tissues, is predisposed to damage. The ‘gene expression’ hypothesis proposes in contrast that Lamin A/C is important regulators of tissue-specific gene expression. There are now evidences to support each hypothesis, since novel emergent concepts propose that the structural weakness/stiffness of the NE, regulated by Lamin A/C amount in NE, can ‘consequently’ alter gene expression. The latest hypothesis suggests a deep interplay between mechanosensitive signalling pathways and transcriptional changes, both involving Lamin A/C. Cells structurally adapt to tissue environments that are continuously subjected to mechanical stress, such as heart and muscles, regulating Lamin A/C expression in the NE. Increased cell tension reduces the turnover of Lamin A/C in the NE and stimulates synthesis of more Lamin A/C. Accumulated Lamin A/C

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could physically reinforce the NE, which would stabilise interactions between chromatin and the nuclear lamina and protect the cell to subsequent nuclear distortions that might otherwise occur in a high-tension tissue environment. Moreover, Lamin A/C expression is accompanied with the activation of SRF pathway controlling expression of structural genes involved in conferring the myogenic phenotype, such as sarcomeric proteins. Finally, the cardiomyocytes senescence seems to be turned on by a significant decrease in Lamin A/C expression in the NE and an increase in Lamin A/C fragmentation. In conclusion, the recent findings clearly confers to Lamin A/C a pivotal role in the all cell life cycle and function of cardiomyocytes suggesting novel considerations on the possible therapeutic intervention for laminopathies.

Future perspectives In both humans and animal models, LMNA mutations cause DCM characterised by AV conduction disorders. Compared with other forms of familial cardiomyopathy, mutations in LMNA are responsible for a more aggressive clinical course due to a high rate of malignant ventricular arrhythmias and end-stage heart failure, and are associated with a higher cardiac disease penetrance, which is almost complete when carriers age (Taylor et al., 2003; van Rijsingen et al., 2012). These manifestations correlate with the postulated crucial role of Lamin A in the cardiomyocytes differentiation, function and senescence, as reviewed in this manuscript. However, the features of specific LMNA mutations still leave open questions on other possible roles and pathways in which this nuclear protein could be involved. Interestingly, the study of a large cohort of LMNA mutation carriers demonstrated that men have a worse prognosis due to a high prevalence of malignant ventricular arrhythmias and end-stage heart failure despite a similar age of onset, suggesting gender difference in the clinical phenotypes in LMNA-dependent DCM (van Rijsingen et al., 2013). Of note, the same gender correlation was found in the LmnaH222P/H222P mice model, displaying males more prominent abnormalities in


cardiac function than females, and dying significantly earlier than female mice (Arimura et al., 2013). In the hearts of these mice, an increased expression and nuclear accumulation of androgen receptors (AR), and a testosterone-induced nuclear translocation of four-and-a-half LIM protein-2 (FHL2) and SRF, was reported. Moreover, in vivo studies revealed the adverse effect of testosterone in the cardiac function and pathological changes in the heart of these mice (Arimura et al., 2013), suggesting that the presence of both testosterone and AR played pivotal roles in the cardiac phenotype of LmnaH222P/H222P mice. Interestingly, no gender difference was observed in other LMNA mutations knock-in mouse models and in a quite large cohort of humans carrying LMNA mutated variants, leaving not resolved how specific LMNA mutations lead to the cardiomyocyte-specific nuclear accumulation of AR and the association with gender differences thus ultimately suggesting that the regulation of this nuclear protein requires still further investigation.

Acknowledgement This work was supported by Fondo per gli Investimenti della Ricerca di Base-Rete Nazionale di Proteomica (RBRN07BMCT 009). Conflict of interest statement The authors have declared no conflict of interest.

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Received: 22 April 2014; Accepted: 16 July 2014; Accepted article online: 23 July 2014

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