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Background: LMNA genotype-positive patients have high risk of experiencing life-threatening ventricular tachyarrhythmias (VTAs). The LMNA-risk VTA calculator published in 2019 has not been externally validated. Objective: The purpose of this study was to validate the LMNA-risk VTA calculator. Methods: We included LMNA genotype-positive patients without previous VTAs from 2 large Scandinavian centers. Patients underwent electrocardiography, 24-hour Holter monitoring, and echocardiographic examinations at baseline and repeatedly during follow-up. Validation of the LMNA-risk VTA calculator was performed using Harrell's C-statistic derived from multivariable Cox regression analysis. Results: We included 118 patients (age 37 years [IQR 27–49 years]; 39 [33%] probands; 65 [55%] women; 100 [85%] with non-missense LMNA variants). Twenty-three patients (19%) experienced VTA during 6.1 years (interquartile range 3.0–9.1 years) follow-up, resulting in 3.0% (95% confidence interval 2.0%–4.5%) yearly incidence rate. Atrioventricular block and reduced left ventricular ejection fraction were independent predictors of VTAs, while nonsustained ventricular tachycardia, male sex, and non-missense LMNA variants were not. The LMNA-risk VTA calculator showed 83% sensitivity and 26% specificity for identifying patients with VTAs during the coming 5 years, and a Harrell's C-statistic of 0.85, when applying ≥7% predicted 5-year VTA risk as threshold. The sensitivity increased to 100% when reevaluating risk at the time of last consultation before VTA. The calculator overestimated arrhythmic risk in patients with mild and moderate phenotype, particularly in men. Conclusion: Validation of the LMNA-risk VTA calculator showed high sensitivity for subsequent VTAs, but overestimated arrhythmic risk when using ≥7% predicted 5-year risk as threshold. Frequent reevaluation of risk was necessary to maintain the sensitivity of the model.

Mutations in the LMNA gene (encoding lamin A/C proteins) cause several human cardiac diseases, including dilated cardiomyopathies (LMNA-DCM). The main clinical risks in LMNA-DCM patients are sudden cardiac death and progressive left ventricular ejection fraction deterioration, and therefore most human and animal studies have sought to define the mechanisms through which LMNA mutations provoke cardiac alterations, with a particular focus on cardiomyocytes. To investigate if LMNA mutations also cause vascular alterations that might contribute to the etiopathogenesis of LMNA-DCM, we generated and characterized Lmnaflox/floxSM22αCre mice, which constitutively lack lamin A/C in vascular smooth muscle cells (VSMCs), cardiac fibroblasts, and cardiomyocytes. Like mice with whole body or cardiomyocyte-specific lamin A/C ablation, Lmnaflox/floxSM22αCre mice recapitulated the main hallmarks of human LMNA-DCM, including ventricular systolic dysfunction, cardiac conduction defects, cardiac fibrosis, and premature death. These alterations were associated with elevated expression of total and phosphorylated (active) Smad3 and cleaved (active) caspase 3 in the heart. Lmnaflox/floxSM22αCre mice also exhibited perivascular fibrosis in the coronary arteries and a switch of aortic VSMCs from the ‘contractile’ to the ‘synthetic’ phenotype. Ex vivo wire myography in isolated aortic rings revealed impaired maximum contraction capacity and an altered response to vasoconstrictor and vasodilator agents in Lmnaflox/floxSM22αCre mice. To our knowledge, our results provide the first evidence of phenotypic alterations in VSMCs that might contribute significantly to the pathophysiology of some forms of LMNA-DCM. Future work addressing the mechanisms underlying vascular defects in LMNA-DCM may open new therapeutic avenues for these diseases.

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Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase overexpressed in various cancer types that plays a critical role in tumor progression. Accumulating evidence suggests that targeting FAK, either alone or in combination with other agents, may serve as an effective therapeutic strategy for numerous cancers. In addition to retarding proliferation, metastasis, and angiogenesis, FAK inhibition triggers cellular senescence in lung cancer cells. However, the detailed mechanism remains enigmatic. In the present study, we found that FAK inhibition not only elicits DNA-damage signaling but also downregulates enhancer of zeste homolog 2 (EZH2) expression. The manipulation of FAK expression influences EZH2 expression and corresponding signaling in vitro. Immunohistochemistry shows that active FAK signaling corresponds with the activation of the EZH2-mediated signaling cascade in lung-cancer-cells-derived tumor tissues. We also found that ectopic EZH2 expression attenuates FAK-inhibition-induced cellular senescence in lung cancer cells. Our results identify EZH2 as a critical downstream effector of the FAK-mediated anti-senescence pathway. Targeting FAK-EZH2 axis-induced cellular senescence may represent a promising therapeutic strategy for restraining tumor growth.

Heterozygous TREX1 mutations are associated with monogenic familial chilblain lupus and represent a risk factor for developing systemic lupus erythematosus. These interferonopathies originate from chronic type I interferon stimulation due to sensing of inadequately accumulating nucleic acids. We here analysed the composition of dendritic cell (DC) subsets, central stimulators of immune responses, in patients with TREX1 deficiency. We performed single-cell RNA-sequencing of peripheral blood DCs and monocytes from two patients with familial chilblain lupus and heterozygous mutations in TREX1 and from controls. Type I interferon pathway genes were strongly upregulated in patients. Cell frequencies of the myeloid and plasmacytoid DC and of monocyte populations in patients and controls were similar, but we describe a novel DC subpopulation highly enriched in patients: a myeloid DC CD1C+ subpopulation characterized by the expression of LMNA, EMP1 and a type I interferon- stimulated gene profile. The presence of this defined subpopulation was confirmed in a second cohort of patients and controls by flow cytometry, also revealing that an increased percentage of patient’s cells in the subcluster express costimulatory molecules. We identified a novel type I interferon responsive myeloid DC subpopulation, that might be important for the perpetuation of TREX1-induced chilblain lupus and other type I interferonopathies.

Background: Mutations in the Lamin A/C (LMNA) gene are responsible for about 6% of all familial dilated cardiomyopathy (DCM) cases which tend to present at a young age and follow a fulminant course. Methods: We report a 47-year-old DCM patient with severely impaired left ventricular ejection fraction and NYHA functional class IV despite optimal heart failure treatment. Whole-exome sequencing revealed an LMNA E161K missense mutation as the pathogenetic cause for DCM in this patient. We generated a patient-specific LMNA-knock in (LMNA-KI) in vitro model using mES cells. Results: Beta adrenergic stimulation of cardiomyocytes derived from LMNA-KI mES cells resulted in augmented mTOR signaling and increased dysregulation of action potentials, which could be effectively prevented by the mTOR-inhibitor rapamycin. A cardiac biopsy confirmed strong activation of the mTOR-signaling pathway in the patient. An off-label treatment with oral rapamycin was initiated and resulted in an improvement in left ventricular ejection fraction (27.8% to 44.5%), NT-BNP (8120 ng/L to 2210 ng/L) and NYHA functional class. Conclusion: We have successfully generated the first in vitro model to recapitulate a patient-specific LMNA E161K mutation which leads to a severe form of DCM. The model may serve as a template for individualized and specific treatment of heart failure.

Recent technological advancements in the field of chromatin biology have rewritten the textbook on nuclear organization. We now appreciate that the folding of chromatin in the three-dimensional space (i.e. its 3D "architecture") is non-random, hierarchical, and highly complex. While 3D chromatin structure is partially encoded in the primary sequence and thereby broadly conserved across cell types and states, a substantial portion of the genome seems to be dynamic during development or in disease. Moreover, there is growing evidence that at least some of the 3D structure of chromatin is functionally linked to gene regulation, both being modulated by and impacting on multiple nuclear processes (including DNA replication, transcription, and RNA splicing). In recent years, these new concepts have nourished several investigations about the functional role of 3D chromatin topology dynamics in the heart during development and disease. This review aims to provide a comprehensive overview of our current understanding in this field, and to discuss how this knowledge can inform further research as well as clinical practice.

Aims: Cardiac implantable electronic device (CIED) therapy is fundamental to the management of LMNA cardiomyopathy due to the high frequency of atrioventricular block and ventricular tachyarrhythmias. We aimed to define the role of cardiac resynchronization therapy (CRT) in impacting heart failure in LMNA cardiomyopathy. Methods and results: From nine referral centres, LMNA cardiomyopathy patients who underwent CRT with available pre- and post-echocardiograms were identified retrospectively. Factors associated with CRT response were identified (defined as improvement in left ventricular ejection fraction [LVEF] ≥5% 6 months post-implant) and the associated impact on the primary outcome of death, implantation of a left ventricular assist device or cardiac transplantation was assessed. We identified 105 patients (mean age 51 ± 10 years) undergoing CRT, including 70 (67%) who underwent CRT as a CIED upgrade. The mean change in LVEF ∼6 months post-CRT was +4 ± 9%. A CRT response occurred in 40 (38%) patients and was associated with lower baseline LVEF or a high percentage of right ventricular pacing prior to CRT in patients with pre-existing CIED. In patients with a European Society of Cardiology class I guideline indication for CRT, response rates were 61%. A CRT response was evident at thresholds of LVEF ≤45% or percent pacing ≥50%. There was a 1.3 year estimated median difference in event-free survival in those who responded to CRT (p = 0.04). Conclusion: Systolic function improves in patients with LMNA cardiomyopathy who undergo CRT, especially with strong guideline indications for implantation. Post-CRT improvements in LVEF are associated with survival benefits in this population with otherwise limited options.

Significance Barrier-to-autointegration factor (BAF), a highly conserved small protein in metazoans, plays multiple functions during the cell cycle; however, the regulation of BAF function is largely unclear. Here we find that the SUMOylation and de-SUMOylation cycle of BAF regulates these functions. We reveal that BAF is SUMOylated at K6, and that de-SUMOylation at this site is catalyzed by SENP1 and SENP2. SUMOylation of BAF regulates binding to lamin A/C, which is essential for nuclear retention of BAF. SUMOylation of BAF also regulates the interaction of BAF with nuclear DNA. De-SUMOylation facilitates cytoplasmic retention of BAF and loss of nuclear function. Non-SUMOylatable BAF is not efficiently retained in the nucleus, leading to structurally abnormal nuclear formation and DNA replication failure., Barrier-to-autointegration factor (BAF) is a highly conserved protein in metazoans that has multiple functions during the cell cycle. We found that BAF is SUMOylated at K6, and that this modification is essential for its nuclear localization and function, including nuclear integrity maintenance and DNA replication. K6-linked SUMOylation of BAF promotes binding and interaction with lamin A/C to regulate nuclear integrity. K6-linked SUMOylation of BAF also supports BAF binding to DNA and proliferating cell nuclear antigen and regulates DNA replication. SENP1 and SENP2 catalyze the de-SUMOylation of BAF at K6. Disrupting the SUMOylation and de-SUMOylation cycle of BAF at K6 not only disturbs nuclear integrity, but also induces DNA replication failure. Taken together, our findings demonstrate that SUMOylation at K6 is an important regulatory mechanism that governs the nuclear functions of BAF in mammalian cells.

Progressive cardiac conduction defect (PCCD) is an inherited autosomal dominant cardiac disorder characterized by an age‑dependent cardiac electrical conduction block. Several genes have been associated with the genetic pathogenesis of PCCD. The present study aimed to identify the causal mutation of PCCD and to investigate the association between genotype and phenotype in a Chinese family with PCCD. A total of 39 family members were included in the present study. All subjects participated in physical, biochemical, electrocardiography and echocardiography examinations. Whole‑exome sequencing was performed for four individuals from the same generation, including three patients with PCCD and one normal control with no cardiovascular disease. Sanger sequencing and in silico analysis were used to identify the causal mutation. Whole‑exome sequencing and variant identification revealed a candidate nonsense mutation (c.1443C>A, p.Tyr481*) in lamin A/C (LMNA). The mutation was identified in seven patients (including the proband) and two asymptomatic mutation carriers, but it was not detected in 100 control subjects of matched ancestry. Clinical examinations identified typical symptoms in patients with PCCD, including bradycardia and various types of conduction defect, and excluded other phenotypes related to the LMNA mutation. The genotype and phenotype were co‑associated among all participants. In the present study, the c.1443C>A mutation in the LMNA gene was identified as a potential cause of PCCD. In silico analysis predicted that the identified mutation was damaging through its effect on the lamin tail domain of LMNA. From the present study, it could be suggested that genetic screening and family counseling, early pacemaker implantation or a sudden death in the family may be essential for risk stratification and treatment of patients with PCCD.

Graphic abstract

Segmental progeroid syndromes are groups of genetic disorders with multiple features resembling accelerated aging. The International Registry of Werner Syndrome (Seattle, WA) recruits pedigrees of progeroid syndromes from all over the world. We identified two novel LMNA mutations, p.Asp300Gly in a patient from Myanmar, and p.Asn466Lys, in a patient from Greece. Both were referred to our Registry for the genetic diagnosis because of the accelerated aged-appearance and cardiac complications. LMNA mutations are the second most common genetic cause of progeroid syndromes after WRN mutations in our Registry. As the next generation sequencing becomes readily available, we expect to identify more cases of rare genetic diseases in the developing countries. Keywords: Lamin A/C, atypical Werner syndrome, progeroid syndrome, medical genetics, human

Decades of studies have established that nuclear lamin polymers form the nuclear lamina, a protein meshwork that supports the nuclear envelope structure and tethers heterochromatin to the nuclear periphery. Much less is known about unpolymerized nuclear lamins in the nuclear interior, some of which are now known to undergo specific phosphorylation. A recent finding that phosphorylated lamins bind gene enhancer regions offers a new hypothesis that lamin phosphorylation may influence transcriptional regulation in the nuclear interior. In this review, we discuss the regulation, localization, and functions of phosphorylated lamins. We summarize kinases that phosphorylate lamins in a variety of biological contexts. Our discussion extends to laminopathies, a spectrum of degenerative disorders caused by lamin gene mutations, such as cardiomyopathies and progeria. We compare the prevailing hypothesis for laminopathy pathogenesis based on lamins’ function at the nuclear lamina with an emerging hypothesis based on phosphorylated lamins’ function in the nuclear interior.

Background and aims: Chromatin interacts with the nuclear lamina (NL) via lamina associated domains (LADs). Nuclear lamin A/C is one of the main candidates for establishing this interaction, but the accurate molecular mechanisms behind it remain unclear. Importantly, Lamin A/C is known to have mechanosensitive properties; transduction of mechanical force to the nucleus is crucial for embryonic development and involved with cancer metastasis. Studying whether the force-induced effects in chromatin are conveyed via lamin A/C may thus explain features of genome organization and provide important clues for understanding the mechanisms of cancer progression. Dam-identification (DamID) is a widely used method for detection of lamin associated LADs, however, its use in epithelial cells has remained unestablished. The aims of this thesis were (1) to describe the intranuclear distribution of lamin A/C and chromatin in epithelial cells, (2) to construct an epithelial cell line with m6a-tracer-DamID system enabling detection and characterization of lamin A/C-interacting LADs, and finally, (3) to observe the role of actin mediated mechanical tension in lamin A/C -mediated LAD organization. Methods: Madin Darby canine kidney type II (MDCK II) cells were used as an epithelial cell model. DamID system was constructed by co-transfecting the system components to the cells. To visualize intranuclear structures in this cell line, immunolabeling and fixed cell imaging with laser scanning confocal microscopy was used. Actin cytoskeleton was disrupted with cytochalasin D treatment to assess the effects of actin mediated force transduction to epithelial cell LADs. Obtained data tested using statistical methods to assess the amplitude and plausibility of the data. Results: Construction of the m6a-tracer-DamID cell line was successful. Analysis revealed LADs in the vicinity of endogenous lamin A/C. These LADs were devoid of transcriptional activity marker H3K27ac, suggesting for their transcriptional repression. Enforcing, some LADs in the apical side of the nucleus were involved with heterochromatin associated H3K9me3. Degradation of actin cytoskeleton resulted in alteration of lamin A/C organization, but these changes were not directly followed by changes in the lateral spatial distribution of LADs. However, LADs were detected to partially detach from the nuclear lamina upon actin disruption. Conclusions: Establishment of MDCKII-m6a-tracer-DamID cell line and its successive use in the experiments of this study indicate the compatibility of DamID system in epithelial cells. The findings of this study implicate that the lamin A/C -associated LADs contain transcriptionally repressed chromatin. Moreover, the results show that loss of actin -mediated mechanical tension in the nucleus does not affect the lateral distribution of lamin A/C -associated LADs but might be involved in tethering chromatin to the nuclear envelope. Finally, the analyses suggest that actin- mediated tension is required for the regulation of lamin A/C structural organization in epithelial cells. Tausta ja tavoitteet: Tumalaminan tiedetään olevan vuorovaikutuksessa kromatiinin kanssa, mutta mekanismit jotka ylläpitävät ja säätelevät tätä yhteyttä ovat toistaiseksi tuntemattomat. Tuman sisäpinnalla sijaitseva lamiini A/C on yksi tärkeistä linkeistä solun tukirangan ja kromatiinin välillä. Lamiini A/C:n tiedetään reagoivan solun aistimiin mekaanisiin voimiin, joten on mahdollista, että lamiini A/C olisi vastuussa myös näiden voimien välittämisestä kromatiinille. Tutkimalla lamiini A/C:n kanssa vuorovaikuttavaa kromatiinia pystytään paremmin ymmärtämään tärkeitä mekanismeja solun kehityksessä sekä syöpien leviämisessä. DamID- metodilla on mahdollista tunnistaa tumalaminan kanssa vuorovaikuttavia kromatiinialueita. Tätä tekniikkaa ei kuitenkaan ole aikaisemmin käytetty epiteelisoluissa. Tämän tutkielman tavoitteena oli (1) tutkia lamiini A/C:n ja kromatiinin jakaumaa epiteelisoluissa, (2) rakentaa DamID-systeemin sisältävä epiteelisolulinja ja käyttää sitä lamiini A/C:n kanssa vuorovaikuttavan kromatiinin analysointiin, ja lopuksi (3) tutkia solun tukirangan välittämän mekaanisen voiman vaikutuksia lamiini A/C:hen ja sen kanssa yhteydessä olevaan kromatiiniin. Metodit: Tutkimuksissa käytetty epiteelisolulinja oli Madin Darby canine kidney type II (MDCK II). DamID- systeemi muodostuu kahdesta komponentista, jotka transfektoitiin soluihin. Solun rakenteiden visualisoimiseksi käytettiin solujen immunoleimausta ja konfokaalimikroskopiaa. Aktiinivälitteisen voiman vaikutuksia tarkasteltiin hajottamalla solun tukiranka sytokalasiini D-myrkyllä. Mittausten tulokset analysoitiin käyttäen statistisia menetelmiä. Tulokset: Solulinjan rakentaminen onnistui, ja sillä tehdyt analyysit tunnistivat kromatiinialueita, jotka sijaitsivat hyvin lähellä lamiini A/C:tä. Näiden alueiden huomattiin sisältävän hyvin vähän aktiivista histoniasetylaatio H3K27ac:ta, viitaten vähäiseen transkriptionaaliseen aktiivisuuteen. Kromatiinin hiljennettyyn tilaan viittasi myös tuman yläpinnan osittainen vuorovaikutus heterokromatiinin kanssa tavatun tri-metyloidun H3K9:n kanssa. Lamiini AC:n C-terminaalisen epitoopin huomattiin korreloivan tumalaminan läheisyydessä tavattujen kromatiinialueiden jakauman kanssa. Aktiini-tukirangan hajoaminen sai aikaan muutoksia lamiini A/C:n organisaatiossa, mutta nämä muutokset eivät suoraan vaikuttaneet kromatiiniin jakaumaan tumassa. Kromatiinin huomattiin kuitenkin sijoittuvan kauemmas tuman pinnasta aktiinin depolymerisaation seurauksena. Päätelmät: Onnistunut solulinjan pystytys ja sen avulla tehdyt kokeet viittasivat siihen, että DamID-systeemi on käyttökelpoinen epiteelisoluissa. Saadun tulokset osoittivat, että lamiini A/C:n kanssa vuorovaikuttava kromatiini on transkriptionaalisesti hiljennettyä. Myrkkykokeiden mukaan aktiinivälitteinen mekaaninen voima ei suoraan heijastu kromatiiniin lamiini A/C:n kautta, mutta saattaa olla vastuussa kromatiinin kiinnittymisestä tumakalvolle. Tutkimuksissa selvisi myös, että mekaanisella voimalla on vaikutus Lamiini A/C:n rakenteelliseen organisaatioon epiteelisoluissa.

Mutations in the LMNA gene cause developing of several phenotypes, both with isolated involvement of cardiac, muscle, adipose and bone tissues, and with their combination. The dominance of cardiovascular signs in the clinical performance and false clarity in nosology definition can cause underestimation of subclinical markers of other systems partaking. It leads to an incorrect interpretation of the true disease etiology, failure in genetic diagnostics, and untimely determination of the correct management and prognosis. The article presents clinical cases that demonstrate both the most typical manifestations of laminopathy and rare combinations of symptoms, which represent a certain diagnostic difficulty.

Myotonic dystrophy type 1 (DM1) is a hereditary and multisystemic disease characterized by myotonia, progressive distal muscle weakness and atrophy. The molecular mechanisms underlying this disease are still poorly characterized, although there are some hypotheses that envisage to explain the multisystemic features observed in DM1. An emergent hypothesis is that nuclear envelope (NE) dysfunction may contribute to muscular dystrophies, particularly to DM1. Therefore, the main objective of the present study was to evaluate the nuclear profile of DM1 patient-derived and control fibroblasts and to determine the protein levels and subcellular distribution of relevant NE proteins in these cell lines. Our results demonstrated that DM1 patient-derived fibroblasts exhibited altered intracellular protein levels of lamin A/C, LAP1, SUN1, nesprin-1 and nesprin-2 when compared with the control fibroblasts. In addition, the results showed an altered location of these NE proteins accompanied by the presence of nuclear deformations (blebs, lobes and/or invaginations) and an increased number of nuclear inclusions. Regarding the nuclear profile, DM1 patient-derived fibroblasts had a larger nuclear area and a higher number of deformed nuclei and micronuclei than control-derived fibroblasts. These results reinforce the evidence that NE dysfunction is a highly relevant pathological characteristic observed in DM1.

Summary The human immunodeficiency virus (HIV) enters the nucleus to establish infection, but the role of nuclear envelope proteins in this process is incompletely understood. Inner nuclear transmembrane proteins SUN1 and SUN2 connect nuclear lamins to the cytoskeleton and participate in the DNA damage response (DDR). Increased levels of SUN1 or SUN2 potently restrict HIV infection through an unresolved mechanism. Here, we find that the antiviral activities of SUN1 and SUN2 are distinct. HIV-1 and HIV-2 are preferentially inhibited by SUN1 and SUN2, respectively. We identify DNA damage inducers that stimulate HIV-1 infection and show that SUN1, but not SUN2, neutralizes this effect. Finally, we show that chromatin movements and nuclear rotations are associated with the effects of SUN proteins and Lamin A/C on infection. These results reveal an emerging role of chromatin dynamics and the DDR in the control of HIV infection by structural components of the nuclear envelope.

Besides its structural properties in the nucleoskeleton, Lamin A/C is a mechanosensor protein involved in perceiving the elasticity of the extracellular matrix. In this study we provide evidence about Lamin A/C-mediated regulation of osteosarcoma cell adhesion and spreading on substrates with tissue-specific elasticities. Our working hypothesis is based on the observation that low-aggressive and bone-resident SaOS-2 osteosarcoma cells express high level of Lamin A/C in comparison to highly metastatic, preferentially to the lung, osteosarcoma 143B cells, thereby suggesting a role for Lamin A/C in tumor cell tropism. Specifically, LMNA gene over-expression in 143B cells induced a reduction in tumor cell aggressiveness in comparison to parental cells, with decreased proliferation rate and reduced migration capability. Furthermore, LMNA reintegration into 143B cells changed the adhesion properties of tumor cells, from a preferential tropism toward the 1.5 kPa PDMS substrate (resembling normal lung parenchyma) to the 28 kPa (resembling pre-mineralized bone osteoid matrix). Our study suggests that Lamin A/C expression could be involved in the organ tropism of tumor cells, thereby providing a rationale for further studies focused on the definition of cancer mechanism of metastatization.

Hutchinson‐Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder most notably characterized by cardiovascular disease and premature death from myocardial infarction or stroke. The majority of cases are caused by a de novo single nucleotide mutation in the LMNA gene that activates a cryptic splice donor site, resulting in production of a toxic form of lamin A with a 50 amino acid internal deletion, termed progerin. We previously reported the generation of a transgenic murine model of progeria carrying a human BAC harboring the common mutation, G608G, which in the single‐copy state develops features of HGPS that are limited to the vascular system. Here, we report the phenotype of mice bred to carry two copies of the BAC, which more completely recapitulate the phenotypic features of HGPS in skin, adipose, skeletal, and vascular tissues. We further show that genetic reduction of the mechanistic target of rapamycin (mTOR) significantly extends lifespan in these mice, providing a rationale for pharmacologic inhibition of the mTOR pathway in the treatment of HGPS., Generation of animal models that express the mutant gene product in the context of the human sequence is critical for developing novel therapeutic approaches to treat HGPS. Here we show that transgenic mice harboring two copies of the human LMNA gene containing the G608G mutation recapitulate the human phenotype. Genetic reduction of mTOR in these mice extends lifespan and provides a rationale for clinical trials testing pharmacological inhibition of the mTOR signaling pathway in HGPS patients.

Clinical effects induced by arrhythmogenic cardiomyopathy (ACM) originate from a large spectrum of genetic variations, including the missense mutation of the lamin A/C gene (LMNA), LMNA D192G. The aim of our study was to investigate the biophysical and biomechanical impact of the LMNA D192G mutation on neonatal rat ventricular fibroblasts (NRVF). The main findings in mutated NRVFs were: (i) cytoskeleton disorganization (actin and intermediate filaments), (ii) decreased elasticity of NRVFs, (iii) altered cell–cell adhesion properties, that highlighted a strong effect on cellular communication, in particular on tunneling nanotubes (TNTs). In mutant-expressing fibroblasts, these nanotubes were weakened with altered mechanical properties as shown by atomic force microscopy (AFM) and optical tweezers. These outcomes complement prior investigations on LMNA mutant cardiomyocytes and suggest that the LMNA D192G mutation impacts the biomechanical properties of both cardiomyocytes and cardiac fibroblasts. These observations could explain how this mutation influences cardiac biomechanical pathology and the severity of ACM in LMNA-cardiomyopathy.

A-type lamins are the main structural components of the nucleus, which are mainly localized at the nucleus periphery. First of all, A-type lamins, together with B-type lamins and proteins of the inner nuclear membrane, form a stiff structure—the nuclear lamina. Besides maintaining the nucleus cell shape, A-type lamins play a critical role in many cellular events, such as gene transcription and epigenetic regulation. Nowadays it is clear that lamins play a very important role in determining cell fate decisions. Various mutations in genes encoding A-type lamins lead to damages of different types of tissues in humans, collectively known as laminopathies, and it is clear that A-type lamins are involved in the regulation of cell differentiation and stemness. However, the mechanisms of this regulation remain unclear. In this review, we discuss how A-type lamins can execute their regulatory role in determining the differentiation status of a cell. We have summarized recent data focused on lamin A/C action mechanisms in regulation of cell differentiation and identity development of stem cells of different origin. We also discuss how this knowledge can promote further research toward a deeper understanding of the role of lamin A/C mutations in laminopathies.

Background Mutations in LMNA, encoding lamin A/C, lead to a variety of diseases known as laminopathies including dilated cardiomyopathy (DCM) and skeletal abnormalities. Though previous studies have investigated the dysregulation of gene expression in cells from patients with DCM, the role of epigenetic (gene regulatory) mechanisms, such as DNA methylation, has not been thoroughly investigated. Furthermore, the impact of family-specific LMNA mutations on DNA methylation is unknown. Here, we performed reduced representation bisulfite sequencing on ten pairs of fibroblasts and their induced pluripotent stem cell (iPSC) derivatives from two families with DCM due to distinct LMNA mutations, one of which also induces brachydactyly. Results Family-specific differentially methylated regions (DMRs) were identified by comparing the DNA methylation landscape of patient and control samples. Fibroblast DMRs were found to enrich for distal regulatory features and transcriptionally repressed chromatin and to associate with genes related to phenotypes found in tissues affected by laminopathies. These DMRs, in combination with transcriptome-wide expression data and lamina-associated domain (LAD) organization, revealed the presence of inter-family epimutation hotspots near differentially expressed genes, most of which were located outside LADs redistributed in LMNA-related DCM. Comparison of DMRs found in fibroblasts and iPSCs identified regions where epimutations were persistent across both cell types. Finally, a network of aberrantly methylated disease-associated genes revealed a potential molecular link between pathways involved in bone and heart development. Conclusions Our results identified both shared and mutation-specific laminopathy epimutation landscapes that were consistent with lamin A/C mutation-mediated epigenetic aberrancies that arose in somatic and early developmental cell stages.

LMNA is one of the leading causative genes of genetically inherited dilated cardiomyopathy (DCM). Unlike most DCM-causative genes, which encode sarcomeric or sarcomere-related proteins, LMNA encodes nuclear envelope proteins, lamin A and C, and does not directly associate with contractile function. However, a mutation in this gene could lead to the development of DCM. The molecular mechanism of how LMNA mutation contributes to DCM development remains largely unclear and yet to be elucidated. The objective of this study was to clarify the mechanism of developing DCM caused by LMNA mutation.Methods and Results: We assessed cardiomyocyte phenotypes and characteristics focusing on cell cycle activity in mice with Lmna mutation. Both cell number and cell size were reduced, cardiomyocytes were immature, and cell cycle activity was retarded in Lmna mutant mice at both 5 weeks and 2 years of age. RNA-sequencing and pathway analysis revealed “proliferation of cells” had the most substantial impact on Lmna mutant mice. Cdkn1a, which encodes the cell cycle regulating protein p21, was strongly upregulated in Lmna mutants, and upregulation of p21 was confirmed by Western blot and immunostaining. DNA damage, which is known to upregulate Cdkn1a, was more abundantly detected in Lmna mutant mice. To assess the proliferative capacity of cardiomyocytes, the apex of the neonate mouse heart was resected, and recovery from the insult was observed. A restricted cardiomyocyte proliferating capacity after resecting the apex of the heart was observed in Lmna mutant mice.Conclusions: Our results strongly suggest that loss of lamin function contributes to impaired cell proliferation through cell cycle defects. The inadequate inborn or responsive cell proliferation capacity plays an essential role in developing DCM with LMNA mutation.

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.

The mechanisms by which lamin A/C in CD4+ T-cells control intestinal homeostasis and can cause inflammatory bowel disease (IBD) are unknown. Here, we explore lamin A/C in a mouse model of IBD. Adoptive transfer to Rag1-/- mice of Lmna-/- CD4+ T-cells, which have enhanced regulatory T-cells (Treg) differentiation and function, induced less severe IBD than wild-type T-cells. Lamin A/C deficiency in CD4+ T-cells enhanced transcription of the Treg master regulator FOXP3, thus promoting Treg differentiation, and reduced Th1 polarization, due to epigenetic changes in the Th1 master regulator T-bet. In mesenteric lymph nodes, retinoic acid (RA) released by CD103+ dendritic cells downregulated lamin A/C in CD4+ T-cells, enhancing Treg differentiation. However, non-RA-producing CD103- dendritic cells predominated in peripheral lymph nodes, facilitating lamin A/C expression in CD4+ T-cells and therefore Th1 differentiation. Our findings establish lamin A/C as a key regulator of Th differentiation in physiological conditions and show it as a potential immune-regulatory target in IBD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. This study was supported by grants to PC from the Research Council of Norway, to JMG-G from ISCIII (PI14/00526; PI17/01395; CP11/00145; CPII16/00022; EuroCellNet COST Action CA15214), the Miguel Servet Program and Fundación Ramón Aréces; to VA (RD12/0042/0028 SAF2013-46663-R, SAF2016-79490-R); to FS-M (SAF2017-82886-R; ERC-2011-AdG 294340-GENTRIS, CIBER CARDIOVASCULAR, PIE 13.0004-BIOIMID and CAM-B2017/BMD-3671-INFLAMUNE), to GC and JLP (PI16/00032 and RETICs RD16/0012 RIER), and to JV (BIO2015-67580-P; PRB3, IPT17/0019 - ISCIII-SGEFI/ERDF, ProteoRed) with co-funding from the European Regional Development Fund (ERDF) “Una manera de hacer Europa”. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). RTF is supported by the Fundación Ramón Aréces, VZG by ISCIII, BHF by the Instituto de Investigación Hospital 12 de Octubre (i+12), and JMG-G by the ISCIII Miguel Servet Program, i+12 and Universidad Autónoma de Madrid (UAM). Sí

The identification of the substrate of idiopathic ventricular fibrillation (VF) in the absence of structural heart disease or electrocardiographic changes remains a challenge for patient management and family counseling. The availability of expanded genetic testing panels and clinical exome screening has not led to a significant increase in the identification of genetic causes of these events, as shown by recent reports.1, 2 Indeed, the majority of studies detected only a small number of genetic variants1, 2 that could explain a sudden cardiac arrest event. Additionally, most variants still remain classified as “of unknown significance” and do not provide information useful for mechanistic understanding and cascade screening. Variants of unknown significance (VUS) of particular complex adjudication are those located at, and possibly altering, RNA splicing sites. RNA testing is thus critical in characterizing splicing alterations and could provide more precise insights compared to in silico prediction algorithms alone. We present here a case of idiopathic VF where RNA studies on the patient’s blood cells helped revise the adjudication of a VUS, providing valuable insights and implications for cascade screening.

Atrioventricular (AV) block is a common condition that occurs at all ages, but most often in the elderly.1 Among younger patients, AV block is most often seen as a complication of cardiac surgery or as congenital AV block. In some (but rare) cases, AV block is due to genetic causes. Variants in the lamin A/C gene are associated with AV block, cardiomyopathy, ventricular arrhythmias, muscular dystrophy, and sudden cardiac death.2, 3 Lamin A and lamin C are major structural proteins of the cell nucleus membrane.4 The cellular mechanism causing AV block in patients with lamin A/C gene variants is not fully understood.5 Previous studies have shown that implanting an implantable cardioverter-defibrillator (ICD) in patients with lamin A/C variants complicated by AV block is effective in treating malignant ventricular arrhythmia (MVA), and thereby potentially preventing sudden cardiac death.6, 7 We report how appropriate use of molecular genetic testing in a 35-year-old man with advanced AV block and family history of sudden cardiac death resulted in an upgrade of the patient’s dual-chamber pacemaker to a biventricular ICD (cardiac resynchronization therapy defibrillator [CRT-D]), which most likely saved the patient’s life.

We report a new laminopathy that includes generalized lipoatrophy, insulin-resistant diabetes, micrognathia and biopsy-proven, focal segmental glomerulosclerosis in a female, caused by a de novo heterozygous mutation R133L in the lamin A/C gene (LMNA). We analysed the nuclear morphology and laminar distribution in 3T3-L1 pre-adipocytes overexpressing human wild-type lamin A/C (LMNA WT) or lamin A/C with the R133L mutation (LMNA R133L). We found the nuclear size was varied, nuclear membrane invagination or blebbing, and an irregular A-type lamin meshwork in cells overexpressing LMNA R133L.3T3-L1 pre-adipocyte differentiation into adipocytes was impaired in cells expressing LMNA R133L; contemporaneously, the expression levels of genes associated with adipose tissue self-renewal, including adipogenesis, angiogenesis, and extracellular matrix maintenance, were downregulated. Furthermore, the insulin-signalling pathway was inhibited in LMNA R133L adipocytes. Microarray gene expression profiling showed that the most prominent differences between 3T3-L1 cells expressing wild-type LMNA and LMNA R133L were in genes implicated in metabolic pathways, the cellular response to DNA damage and repair. We thus expand the clinical spectrum of laminopathy and conclude that the LMNA R133L mutation associated with lipodystrophic features and multisystem disorders likely impairs adipocyte renewal and disrupts the expression of genes implicated in the induction and repair of DNA damage.

The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery–Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ8–11 murine model of Emery–Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ8–11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.

Intermediate filaments are major components of the cytoskeleton. Desmin and synemin, cytoplasmic intermediate filament proteins and A-type lamins, nuclear intermediate filament proteins, play key roles in skeletal and cardiac muscle. Desmin, encoded by the DES gene (OMIM *125660) and A-type lamins by the LMNA gene (OMIM *150330), have been involved in striated muscle disorders. Diseases include desmin-related myopathy and cardiomyopathy (desminopathy), which can be manifested with dilated, restrictive, hypertrophic, arrhythmogenic, or even left ventricular non-compaction cardiomyopathy, Emery–Dreifuss Muscular Dystrophy (EDMD2 and EDMD3, due to LMNA mutations), LMNA-related congenital Muscular Dystrophy (L-CMD) and LMNA-linked dilated cardiomyopathy with conduction system defects (CMD1A). Recently, mutations in synemin (SYNM gene, OMIM *606087) have been linked to cardiomyopathy. This review will summarize clinical and molecular aspects of desmin-, lamin- and synemin-related striated muscle disorders with focus on LMNA and DES-associated clinical entities and will suggest pathogenetic hypotheses based on the interplay of desmin and lamin A/C. In healthy muscle, such interplay is responsible for the involvement of this network in mechanosignaling, nuclear positioning and mitochondrial homeostasis, while in disease it is disturbed, leading to myocyte death and activation of inflammation and the associated secretome alterations.

Background: Variants in the LMNA gene, encoding lamins A/C, are responsible for a growing number of diseases, all of which complying with the definition of rare diseases. LMNA-related disorders have a varied phenotypic expression with more than 15 syndromes described, belonging to five phenotypic groups: Muscular Dystrophies, Neuropathies, Cardiomyopathies, Lipodystrophies and Progeroid Syndromes. Overlapping phenotypes are also reported. Linking gene and variants with phenotypic expression, disease mechanisms, and corresponding treatments is particularly challenging in laminopathies. Treatment recommendations are limited, and very few are variant-based. Objective: The Treatabolome initiative aims to provide a shareable dataset of existing variant-specific treatment for rare diseases within the Solve-RD EU project. As part of this project, we gathered evidence of specific treatments for laminopathies via a systematic literature review adopting the FAIR (Findable, Accessible, Interoperable, and Reusable) guidelines for scientific data production. Methods: Treatments for LMNA-related conditions were systematically collected from MEDLINE and Embase bibliographic databases and clinical trial registries (Cochrane Central Registry of Controlled Trials, clinicaltrial.gov and EudraCT). Two investigators extracted and analyzed the literature data independently. The included papers were assessed using the Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence. Results: From the 4783 selected articles by a systematic approach, we identified 78 papers for our final analysis that corresponded to the profile of data defined in the inclusion and exclusion criteria. These papers include 2 guidelines/consensus papers, 4 meta-analyses, 14 single-arm trials, 15 case series, 13 cohort studies, 21 case reports, 8 expert reviews and 1 expert opinion. The treatments were summarized electronically according to significant phenome-genome associations. The specificity of treatments according to the different laminopathic phenotypical presentations is variable. Conclusions: We have extracted Treatabolome-worthy treatment recommendations for patients with different forms of laminopathies based on significant phenome-genome parings. This dataset will be available on the Treatabolome website and, through interoperability, on genetic diagnosis and treatment support tools like the RD-Connect’s Genome Phenome Analysis Platform.

Introduction: Cardiac involvement in patients with muscular dystrophy associated with Lamin A/C mutations (LMNA) is characterized by atrioventricular conduction abnormalities and life-threatening cardiac arrhythmias. Little is known about cardiac involvement in patients with emerin mutation (EMD). The aim of our study was to describe and compare the prevalence and time distribution of cardiac arrhythmias at extended follow-up. Patients and methods: 45 consecutive patients affected by muscular dystrophy associated to laminopathy or emerinopathy were examined. All patients underwent clinical evaluation, 12-lead surface electrocardiogram (ECG), 24 h electrocardiographic monitoring, and cardiac implanted device interrogation. Results: At the end of 11 (5.0–16.6) years of follow-up, 89% of the patients showed cardiac arrhythmias. The most prevalent was atrial standstill (AS) (31%), followed by atrial fibrillation/flutter (AF/Afl) (29%) and ventricular tachycardia (22%). EMD patients presented more frequently AF/AFl compared to LMNA (50% vs. 20%, p = 0.06). Half of the EMD patients presented with AS, whilst there was no occurrence of such in the LMNA (p = 0.001). Ventricular arrhythmias were found in 60% of patients with laminopathy compared to 3% in patients with emerinopathy (p &lt, 0.001). The age of AVB occurrence was higher in the LMNA group (32.8 +/− 10.6 vs. 25.1 +/− 9.1, p = 0.03). Conclusions: Atrial arrhythmias are common findings in patients with muscular dystrophy associated with EMD/LMNA mutations, however, they occurred earlier in EMD patients. Ventricular arrhythmias were very common (60%) in LMNA and occurred definitely earlier compared to the EMD group.

Nuclear lamina components have long been regarded as scaffolding proteins, forming a dense fibrillar structure necessary for the maintenance of the nucleus shape in all the animal kingdom. More recently, mutations, aberrant localisation and deregulation of these proteins have been linked to several diseases, including cancer. Using publicly available data we found that the increased expression levels of the nuclear protein Lamin A/C correlate with a reduced overall survival in The Cancer Genome Atlas Research Network (TCGA) patients affected by glioblastoma multiforme (GBM). We show that the expression of the LMNA gene is linked to the enrichment of cancer-related pathways, particularly pathways related to cell adhesion and cell migration. Mimicking the modulation of LMNA in a GBM preclinical cancer model, we confirmed both in vitro and in vivo that the increased expression of LMNA is associated with an increased aggressiveness and tumorigenicity. In addition, delving into the possible mechanism behind LMNA-induced GBM aggressiveness and tumorigenicity, we found that the mTORC2 component, Rictor, plays a central role in mediating these effects.

Ο πυρήνας αποτελεί το χαρακτηριστικότερο κυτταρικό οργανίδιο και ένα από τα λίγα που φέρουν διπλή μεμβράνη. Η ετερογένεια που παρατηρείται μεταξύ των μεμβρανών του πυρηνικού φακέλου οφείλεται κυρίως στις πρωτεΐνες που διαθέτουν. Συγκεκριμένα, η εσωτερική πυρηνική μεμβράνη αποτελεί ένα εξαιρετικό εξειδικευμένο μεμβρανικό διαμέρισμα, όπου εντοπίζονται πρωτεΐνες που αλληλεπιδρούν ποικιλοτρόπως με τη χρωματίνη και τις λαμίνες. Οι τελευταίες είναι ενδιάμεσα ινίδια που σχηματίζουν δίκτυα στην πυρηνοπλασματική πλευρά της εσωτερικής πυρηνικής μεμβράνης τα οποία ονομάζονται πυρηνική λάμινα και συμμετέχουν σε πολλές σημαντικές διεργασίες του DNA. Το μεγαλύτερο μέρος του πυρηνοπλάσματος καταλαμβάνουν οι ευχρωματινικές και ετεροχρωματινικές αλληλουχίες του DNA. Η τοποθέτησή τους εντός του πυρήνα είναι πολύ σημαντική για τη φυσιολογική λειτουργία του κυττάρου και ρυθμίζεται σε μεγάλο βαθμό από την πυρηνική περιφέρεια. Συγκεκριμένα, η εκτεταμένη πρόσδεση της χρωματίνης στον πυρηνικό φάκελο καθορίζει την συμβατική πυρηνική αρχιτεκτονική, στην οποία η ετεροχρωματίνη εντοπίζεται κυρίως στα όρια του πυρήνα και η ευχρωματίνη στο εσωτερικό του. Επιπλέον, η περιφέρεια του πυρήνα ρυθμίζει σημαντικά το επιγενετικό τοπίο και την συντονισμένη γονιδιακή αποσιώπηση, ιδίως στην διαδικασία της διαφοροποίησης. Μελέτες σε πυρήνες με διαταραγμένη την συμβατική χρωματινική δομή έχουν αναδείξει δύο κύριες πρωτεΐνες πρόσεδεσης της χρωματίνης στην περιφέρεια του πυρήνα, τον LBR και την Lamin A/C. Στην παρούσα εργασία το ενδιαφέρον επικεντρώθηκε στις δύο αυτές πρωτεΐνες και μελετήθηκαν οι απαλοιφές τους τόσο μεμονωμένα όσο και συνδυαστικά σε κύτταρα NIH/3T3. Οι περισσότερες μεταβολές, τόσο στην κατανομή των πρωτεϊνών του πυρηνικού φακέλου όσο και στο χρωματινικό τοπίο, παρατηρήθηκαν στην ταυτόχρονη απουσία των Lamin A/C και LBR. Η συνδυαστική έλλειψη τους από την πυρηνική περιφέρεια συνδυάστηκε με ασυνέχεια στην κατανομή πρωτεϊνών του πυρηνικού φακέλου και μεταβολή της συμβατικής πυρηνικής αρχιτεκτονικής σε διαφοροποιημένα κύτταρα. Παρόλο που η πυρηνική περιφέρεια είναι από τα πολυπλοκότερα μέρη του κυττάρου, αποτελεί πρόκληση η διευκρίνιση των ρόλων της στην χρωματινική δομή και είναι πολύ πιθανό οι πρωτεΐνες των LBR και Lamin A/C να συμμετέχουν καθοριστικά σε αυτή. The nucleus is the most characteristic cellular organelle and one of the few that bear a double membrane. The observed heterogeneity between the membranes of the nuclear envelope is mostly due to their proteins. In particular, the inner nuclear membrane is an extremely specialized membrane compartment, which includes proteins that interact in various ways with chromatin and lamins. The latter are intermediate filaments that form networks in the nucleoplasmic side of the inner nuclear membrane, called nuclear lamina and are involved in many important DNA processes. Most of the nucleoplasm is occupied by euchromatic and heterochromatic DNA sequences. Their placement inside the nucleus is very important for normal cell function and is largely regulated by the nuclear periphery. Particularly, the extensive chromatin binding to the nuclear envelope determines the conventional nuclear architecture, in which heterochromatin is located mostly at the boundaries of the nucleus and the euchromatin within it. Moreover, the nuclear periphery significantly regulates the epigenetic landscape and coordinated genome silencing, especially during the differentiation process. Studies in nucleι with disrupted conventional chromatin structure have identified two major tethers of chromatin in the nuclear periphery, LBR and Lamin A/C. This current study focuses on these two proteins and their deletion studied both individually and in combination, in NIH/3T3 cells. Most of the changes in the distribution of the nuclear envelope proteins and in the chromatin landscape, were observed in the simultaneous absence of Lamin A/C and LBR. Their combined lack from the nuclear periphery was associated with discontinuity in the localization of proteins in the nuclear envelope and variation of conventional nuclear architecture in differentiated cells. Although the nuclear periphery is one of the most complex parts of the cell, it is challenging to clarify its’ roles in chromatin structure, and it is highly likely that the LBR and Lamin A/C participate crucially in that. 181 σ.

Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely \(\it LMNA\) (lamin A/C), \(\it RBM20\) (RNA binding motif protein 20), \(\it TTN\) (titin) and \(\it PKP2\) (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in \(\it RBM20\) and \(\it TTN\) as the splice factor RBM20 targets amongst other genes \(\it TTN\), leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in \(\it LMNA\) is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in \(\it PKP2\) lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies.

The US3 serine/threonine protein kinase is conserved among the alphaherpesvirus family and represents an important virulence factor. US3 plays a role in viral nuclear egress, induces dramatic alterations of the cytoskeleton, represses apoptosis, enhances gene expression and modulates the immune response. Although several substrates of US3 have been identified, an unbiased screen to identify US3 phosphorylation targets has not yet been described. Here, we perform a shotgun and phosphoproteomics analysis of cells expressing the US3 protein of pseudorabies virus (PRV) to identify US3 phosphorylation targets in an unbiased way. We identified several cellular proteins that are differentially phosphorylated upon US3 expression and validated the phosphorylation of lamin A/C at serine 404, both in US3-transfected and PRV-infected cells. These results provide new insights into the signaling network of the US3 protein kinase and may serve as a basis for future research into the role of the US3 protein in the viral replication cycle.

The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery–Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ8–11 murine model of Emery–Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ8–11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.

Our understanding of the tumorigenesis of classical Hodgkin lymphoma (cHL) and the formation of Reed–Sternberg cells (RS-cells) has evolved drastically in the last decades. More recently, a better characterization of the signaling pathways and the cellular interactions at play have paved the way for new targeted therapy in the hopes of improving outcomes. However, important gaps in knowledge remain that may hold the key for significant changes of paradigm in this lymphoma. Here, we discuss the past, present, and future of cHL, and review in detail the more recent discoveries pertaining to genetic instability, anti-apoptotic signaling pathways, the tumoral microenvironment, and host-immune system evasion in cHL.

Mutations of Lamin A/C gene (LMNA) cause laminopathies, a group of disorders associated with a wide spectrum of clinically distinct phenotypes, affecting different tissues and organs. Heart involvement is frequent and leads to cardiolaminopathy LMNA-dependent cardiomyopathy (LMNA-CMP), a form of dilated cardiomyopathy (DCM) typically associated with conduction disorders and arrhythmias, that can manifest either as an isolated event or as part of a multisystem phenotype. Despite the recent clinical and molecular developments in the field, there is still lack of knowledge linking specific LMNA gene mutations to the distinct clinical manifestations. Indeed, the severity and progression of the disease have marked interindividual variability, even amongst members of the same family. Studies conducted so far have described Lamin A/C proteins involved in diverse biological processes, that span from a structural role in the nucleus to the regulation of response to mechanical stress and gene expression, proposing various mechanistic hypotheses. However, none of those is per se able to fully justify functional and clinical phenotypes of LMNA-CMP; therefore, the role of Lamin A/C in cardiac pathophysiology still represents an open question. In this review we provide an update on the state-of-the-art studies on cardiolaminopathy, in the attempt to draw a line connecting molecular mechanisms to clinical manifestations. While investigators in this field still wonder about a clear genotype/phenotype correlation in LMNA-CMP, our intent here is to recapitulate common mechanistic hypotheses that link different mutations to similar clinical presentations.

Aims Hutchinson–Gilford progeria syndrome (HGPS) is an ultrarare laminopathy caused by expression of progerin, a lamin A variant, also present at low levels in non-HGPS individuals. HGPS patients age and die prematurely, predominantly from cardiovascular complications. Progerin-induced cardiac repolarization defects have been described previously, although the underlying mechanisms are unknown. Methods and results We conducted studies in heart tissue from progerin-expressing LmnaG609G/G609G (G609G) mice, including microscopy, intracellular calcium dynamics, patch-clamping, in vivo magnetic resonance imaging, and electrocardiography. G609G mouse cardiomyocytes showed tubulin-cytoskeleton disorganization, t-tubular system disruption, sarcomere shortening, altered excitation–contraction coupling, and reductions in ventricular thickening and cardiac index. G609G mice exhibited severe bradycardia, and significant alterations of atrio-ventricular conduction and repolarization. Most importantly, 50% of G609G mice had altered heart rate variability, and sinoatrial block, both significant signs of premature cardiac aging. G609G cardiomyocytes had electrophysiological alterations, which resulted in an elevated action potential plateau and early afterdepolarization bursting, reflecting slower sodium current inactivation and long Ca+2 transient duration, which may also help explain the mild QT prolongation in some HGPS patients. Chronic treatment with low-dose paclitaxel ameliorated structural and functional alterations in G609G hearts. Conclusions Our results demonstrate that tubulin-cytoskeleton disorganization in progerin-expressing cardiomyocytes causes structural, cardiac conduction, and excitation–contraction coupling defects, all of which can be partially corrected by chronic treatment with low dose paclitaxel., Graphical Abstract

Although mutations in the Lamin A/C gene (LMNA) cause a variety of devastating diseases, the pathological mechanism is often unknown. Lamin A/C proteins play a crucial role in forming a meshwork under the nuclear membrane, providing the nucleus with mechanical integrity and interacting with other proteins for gene regulation. Most LMNA mutations result in heart diseases, including some types that primarily have heart disease as the main pathology. In this study, we used cells from patients with different LMNA mutations that primarily lead to heart disease. Indeed, it is a mystery why a mutation to the protein in every nucleus of the body manifests as a disease of primarily the heart in these patients. Here, we aimed to investigate if strains mimicking those within the myocardial environment are sufficient to cause differences in cells with and without the LMNA mutation. To test this, a stretcher device was used to induce cyclic strain upon cells, and viability/proliferation, cytoskeleton and extracellular matrix organization, and nuclear morphology were quantified. The properties of cells with Hutchinson-Gilford progeria syndrome (HGPS) were found to be significantly different from all other cell lines and were mostly in line with previous findings. However, the properties of cells from patients who primarily had heart diseases were not drastically different when compared to individuals without the LMNA mutation. Our results indicated that cyclic strain alone was insufficient to cause any significant differences that could explain the mechanisms that lead to heart diseases in these patients with LMNA mutations.

Background Pathogenic variants in lamin A/C (LMNA) cause a variety of progeroid disorders including Hutchinson-Gilford progeria syndrome, mandibuloacral dysplasia, and atypical progeroid syndrome. Six families with 11 patients harboring a pathogenic heterozygous LMNA c.1045C>T; p.R349W variant have been previously reported to have partial lipodystrophy, cardiomyopathy, and focal segmental glomerulosclerosis (FSGS), suggesting a distinct progeroid syndrome. Methods We report 6 new patients with a heterozygous LMNA p.R349W variant and review the phenotype of previously reported patients to define their unique characteristics. We also performed functional studies on the skin fibroblasts of a patient to seek the underlying mechanisms of various clinical manifestations. Results Of the total 17 patients, all 14 adults with the heterozygous LMNA p.R349W variant had peculiar lipodystrophy affecting the face, extremities, palms, and soles with variable gain of subcutaneous truncal fat. All of them had proteinuric nephropathy with FSGS documented in 7 of them. Ten developed cardiomyopathy, and 2 of them died early at ages 33 and 45 years. Other common features included premature graying, alopecia, high-pitched voice, micrognathia, hearing loss, and scoliosis. Metabolic complications, including diabetes mellitus, hypertriglyceridemia, and hepatomegaly, were highly prevalent. This variant did not show any abnormal splicing, and no abnormal nuclear morphology was noted in the affected fibroblasts. Conclusions The heterozygous LMNA p.R349W variant in affected individuals has several distinct phenotypic features, and these patients should be classified as having multisystem progeroid syndrome (MSPS). MSPS patients should undergo careful assessment at symptom onset and yearly metabolic, renal, and cardiac evaluation because hyperglycemia, hypertriglyceridemia, FSGS, and cardiomyopathy cause major morbidity and mortality.

BACKGROUND: Laminopathies are genetic diseases caused by mutations in the nuclear lamina. OBJECTIVE: Given the clinical impact of laminopathies, understanding mechanical properties of cells bearing lamin mutations will lead to advancement in the treatment of heart failure. METHODS: Atomic force microscopy (AFM) was used to analyze the viscoelastic behavior of neonatal rat ventricular myocyte cells expressing three human lamin A/C gene (LMNA) mutations. RESULTS: Cell storage modulus is characterized, by two plateaus, one in the low frequency range, a second one at higher frequencies. The loss modulus instead shows a “bell” shape with a relaxation toward fluid properties at lower frequencies. Mutations shift the relaxation to higher frequencies, rendering the networks more solid-like. Is interesting that this increase of mutations stiffness (solid like behavior) is at frequencies around 1 Hz, close to the human heart rate. CONCLUSIONS: These features result from a combination of the properties of cytoskeleton filaments and their temporary cross-linker. Our results substantiate that cross-linked filaments contribute for the most part of the mechanical strength of the cytoskeleton cell studied and the relaxation time is determined by the dissociation dynamics of the cross-linking proteins. The severity of biomechanical defects due to these LMNA mutations correlated with the severity of the clinical phenotype.

International audience; This study details the clinical and cellular phenotypes associated with two missense heterozygous mutations in LMNA, c.1745G>T p.(Arg582Leu), and c.1892G>A p.(Gly631Asp), in two patients with early onset of diabetes mellitus, hypertriglyceridemia and non-alcoholic fatty liver disease. In these two patients, subcutaneous adipose tissue was persistent, at least on the abdomen, and the serum leptin level remained in the normal range. Cellular studies showed elevated nuclear anomalies, an accelerated senescence rate and a decrease of replication capacity in patient cells. In cellular models, the overexpression of mutated prelamin A phenocopied misshapen nuclei, while the partial reduction of lamin A expression in patient cells significantly improved nuclear morphology. Altogether, these results suggest a link between lamin A mutant expression and senescence associated phenotypes. Transcriptome analysis of the whole subcutaneous adipose tissue from the two patients and three controls, paired for age and sex using RNA sequencing, showed the up regulation of genes implicated in immunity and the down regulation of genes involved in development and cell differentiation in patient adipose tissue. Therefore, our results suggest that some mutations in LMNA are associated with severe metabolic phenotypes without subcutaneous lipoatrophy, and are associated with nuclear misshaping.

This study details the clinical and cellular phenotypes associated with two missense heterozygous mutations in LMNA, c.1745G &gt, T p.(Arg582Leu), and c.1892G&gt, A p.(Gly631Asp), in two patients with early onset of diabetes mellitus, hypertriglyceridemia and non-alcoholic fatty liver disease. In these two patients, subcutaneous adipose tissue was persistent, at least on the abdomen, and the serum leptin level remained in the normal range. Cellular studies showed elevated nuclear anomalies, an accelerated senescence rate and a decrease of replication capacity in patient cells. In cellular models, the overexpression of mutated prelamin A phenocopied misshapen nuclei, while the partial reduction of lamin A expression in patient cells significantly improved nuclear morphology. Altogether, these results suggest a link between lamin A mutant expression and senescence associated phenotypes. Transcriptome analysis of the whole subcutaneous adipose tissue from the two patients and three controls, paired for age and sex using RNA sequencing, showed the up regulation of genes implicated in immunity and the down regulation of genes involved in development and cell differentiation in patient adipose tissue. Therefore, our results suggest that some mutations in LMNA are associated with severe metabolic phenotypes without subcutaneous lipoatrophy, and are associated with nuclear misshaping.

Background Lamin A/C cardiomyopathy is a malignant and highly penetrant inheritable cardiomyopathy. Competitive sports have been associated with adverse events in these patients, but data on recreational exercise are lacking. We aimed to explore associations between exercise exposure and disease severity in patients with lamin A/C genotype. Methods and Results Lamin A/C genotype positive patients answered a questionnaire on exercise habits from age 7 years until genetic diagnosis. We recorded exercise hours >3 metabolic equivalents and calculated cumulative lifetime exercise. Patients were grouped in active or sedate based on lifetime exercise hours above or below median. We performed echocardiography, 12‐lead ECG, Holter monitoring, and biomarkers including NT‐proBNP (N‐terminal pro‐B‐type natriuretic peptide). We defined left ventricular ejection fraction

LMNA encodes for Lamin A/C, type V intermediate filaments that polymerize under the inner nuclear membrane to form the nuclear lamina. A small fraction of Lamin A/C, less polymerized, is also found in the nucleoplasm. Lamin A/C functions include roles in nuclear resistance to mechanical stress and gene regulation. LMNA mutations are responsible for a wide variety of pathologies, including Emery–Dreifuss (EDMD) and LMNA-related congenital muscular dystrophies (L-CMD) without clear genotype–phenotype correlations. Both diseases presented with striated muscle disorders although L-CMD symptoms appear much earlier and are more severe. Seeking for pathomechanical differences to explain the severity of L-CMD mutations, we performed an in silico analysis of the UMD-LMNA database and found that L-CMD mutations mainly affect residues involved in Lamin dimer and tetramer stability. In line with this, we found increased nucleoplasmic Lamin A/C in L-CMD patient fibroblasts and mouse myoblasts compared to the control and EDMD. L-CMD myoblasts show differentiation defects linked to their inability to upregulate muscle specific nuclear envelope (NE) proteins expression. NE proteins were mislocalized, leading to misshapen nuclei. We conclude that these defects are due to both the absence of Lamin A/C from the nuclear lamina and its maintenance in the nucleoplasm of myotubes.

Given the clinical effect of laminopathies, understanding lamin mechanical properties will benefit the treatment of heart failure. Here we report a mechano-dynamic study of LMNA mutations in neonatal rat ventricular myocytes (NRVM) using single cell spectroscopy with Atomic Force Microscopy (AFM) and measured changes in beating force, frequency and contractile amplitude of selected mutant-expressing cells within cell clusters. Furthermore, since beat-to-beat variations can provide clues on the origin of arrhythmias, we analyzed the beating rate variability using a time-domain method which provides a Poincaré plot. Data were further correlated to cell phenotypes. Immunofluorescence and calcium imaging analysis showed that mutant lamin changed NRVMs beating force and frequency. Additionally, we noted an altered microtubule network organization with shorter filament length, and defective hemichannel membrane localization (Connexin 43). These data highlight the interconnection between nucleoskeleton, cytoskeleton and sarcolemmal structures, and the transcellular consequences of mutant lamin protein in the pathogenesis of the cardiac laminopathies., Biological sciences; Cardiology; Biomechanics; Cytoskeleton; Mechanical property; Membrane; Lamin A/C; Cardiomyopathy; Atomic force microscopy; Cardiomyocytes; Beating; Cx43