LMNA Research Articles

FRI458 Rare Pathogenic Missense Variants in LMNA Identified in Women with Polycystic Ovary Syndrome (PCOS)

Abstract Disclosure: R. Bauer: None. L. Gorsic: None. R.S. Legro: None. M.G. Hayes: None. M. Urbanek: None. Background: Polycystic ovary syndrome (PCOS) is the most common form of anovulatory infertility among reproductive age women. In addition to experiencing reproductive symptoms, women with PCOS are at elevated risk of developing obesity, insulin resistance (IR), and type 2 diabetes. Familial partial lipodystrophy type 2 (FPLD2) is a disorder of lipid storage and insulin resistance caused by dominant missense alleles in the gene encoding the intermediate filaments lamin A/C (LMNA). Women with this Mendelian disorder of IR also experience symptoms of PCOS such as amenorrhea and hyperandrogenism. We, therefore, hypothesize that genetic variation in LMNA also contributes to PCOS, which is a common form of IR. In other words, we hypothesize that PCOS falls into the phenotypic spectrum of disorders caused by variation in LMNA.Objective: We aim to identify and evaluate variation in LMNA that underlies PCOS pathogenesis. Methods: To test our hypothesis, we sequenced the LMNA gene in 602 women with PCOS and 125 reproductively healthy women. We comprehensively screened LMNA for genetic variation that is likely to alter the lamin A/C proteins, including missense, nonsense, splicing or frameshift variants. We identified 7 missense variants in 8 cases and no variants in reproductively healthy controls (χ2= 3.1, p=0.081, OR > 1.78, with study controls; χ2= 46.8, p<1x10-8, OR= 8.5 with gnomAD non-Finnish European cohort population controls). To determine which of these variants are pathogenic, we systematically evaluated them according to criteria outlined by the American College of Medical Genetics (ACMG). These guidelines evaluate pathogenicity of variants through multiple lines of evidence, including population data, computational predictions, and functional studies. Results: We identified 5 pathogenic variants in LMNA and 2 that are likely pathogenic. When assessed individually, 6 of the 7 variants are significantly enriched in our cohort when compared to gnomAD non-Finnish European population controls (OR > 5.0). All of the LMNA variants are likely damaging as predicted by 3 computational methods: CADD score ≥ 15, FATHMM prediction of “Damaging”, and MutationTaster classification as “disease causing.” Additionally, many of the variants have previously been identified in individuals with lipodystrophy. Conclusion: Together with previous identification of LMNA variants in women with PCOS by our lab (Urbanek et al. 2009 JCEM) and others (Crespo et al. 2022 JES), this work further establishes LMNA variation as a pathogenic mechanism for PCOS. Presentation Date: Friday, June 16, 2023

OR11-01 A New Model for Estrogen Receptor-alpha Antagonist Mechanism of Action

Abstract Disclosure: J.C. Nwachukwu: None. J.W. Njeri: None. T. Venables: None. C. Seath: None. M.E. Pipkin: None. Y. Hou: None. B.S. Katzenellenbogen: None. J.A. Katzenellenbogen: None. K.W. Nettles: None. We recently developed Dual Mechanism Estrogen Receptor-α (ERα) Inhibitors (DMERI) with more antagonist efficacy than selective ERα modulators (SERMs) such as tamoxifen, and selective ERα degraders (SERDs) such as fulvestrant in certain breast cancer models. Current models of anti-estrogen action are largely based on genomics studies in growth arrested cells, where the effects primarily reversed estradiol-mediated gene expression. In that context, most ER antagonists, especially the SERDs, show very little gene activation. We were interested in understanding antagonist mechanism of action in asynchronous cells that were growing in intact medium that had not been charcoal-stripped to remove estradiol and growth factors. RNA-seq of asynchronous MCF-7 cells treated with 4-hydroxytamoxifen, fulvestrant, a SERM-like DMERI, and a SERD-like DMERI showed that all four classes of ligands equally up-regulated and down-regulated differentially expressed genes (DEGs), which included a core set of regulatory networks, as well as ligand-selective gene networks. The number of DEGs predicted efficacy, with tamoxifen regulating few genes, and the SERD-like DMERI regulating the most genes. The SERD-like DMERI also inhibited expression of many more genes that encode cell cycle regulators and key cancer drivers, such as ERα, AKT1, HRAS, and several growth factor receptors. Landscape in silico analysis based on chromatin immunoprecipitation (ChIP)-seq data suggests that recruitment of the ERα MegaTrans complex in collaboration with the oxysterol receptor, LXRα, predicts up-regulation, while cooperation with the retinoblastoma-associated proteins, RB1 and RBL1, predicts down-regulation of target genes. APEX2-mediated proximity labeling in asynchronous cells revealed that fulvestrant and the SERD-like DMERI labeled both coactivators and corepressors. Both compounds also increase labeling of STAG1 and other cohesin subunits that coordinate chromatin looping, while also associating with the nuclear matrix protein Lamin B1 and heterochromatin-associated proteins. These data suggest that antagonists have dual roles in genome organization. An shRNAmiR screen targeting over 300 chromatin regulatory factors revealed networks of shared and exclusive coregulators used by antiestrogens, including the heterochromatin protein, CBX1, which was exclusively required for growth inhibition by fulvestrant, and subunits of histone acetylase and methylase complexes such as KMT2A (the catalytic subunit of the MLL1 complex), which were broadly required. Thus, antiestrogen-driven growth suppression involves both common and ligand-selective transcriptional regulatory networks through utilization of both coactivators and corepressors. Supported by NIH R01CA220284 and BCRF-083 and BCRF-084 grants. Presentation Date: Friday, June 16, 2023

An alpha-helix variant p.Arg156Pro in LMNA as a cause of hereditary dilated cardiomyopathy: genetics and bioinfomatics exploration

LMNA gene encodes lamin A/C protein which participates in the construction of nuclear lamina, the mutations of LMNA result in a wide variety of diseases known as laminopathies. LMNA-related dilated cardiomyopathy(LMNA-DCM) is one of the more common laminopathy which characterized by progressive heart failure and arrhythmia. However, the mutation features of LMNA-DCM are yet to be elucidated. Herein we described a dilated cardiomyopathy family carrying novel variant c.467G > C(p.Arg156Pro) of LMNA as heterozygous pathogenic variant identified by whole-exome sequencing. With the help of Alphafold2, we predicted mutant protein structure and found an interrupted α-helix region in lamin A/C. In the analysis of 49 confirmed pathogenic missense of laminopathies, Chi-square test showed the DCM phenotype was related to the α-helix region mutation (p < 0.017). After screening the differentially expressed genes (DEGs) in both mice models and human patients in Gene Expression Omnibus database, we found the variation of α-helix-coding region in LMNA caused abnormal transcriptomic features in cell migration, collagen-containing extracellular matrix, and PI3K-Akt signaling pathway. Subsequently we constructed (TF)-mRNA-microRNA (miRNA) regulatory network and identified 7 key genes (FMOD, CYP1B1, CA3, F2RL1, HAPLIN1, SNAP91, and KANSL1) as potential biomarkers or therapeutic targets in LMNA-DCM patients.

STRUCTURAL CHARACTERIZATION OF THE MUTANTS OF LAMINA ROD2 BDOMAIN

LaminAbelongstotypeVintermediatefilamentproteinfamilyandispresentinallmetazoan cells except plants. Lamins provide structural rigidity to the nucleus, tether thechromosomesandmaintainhomeostasisbyplayingdiverserolesinreplication,Transcription,andDNAdamagerepair.Overthelasttwodecadestherehasbeenatremendous impetus in the research involving laminsdue to the fact that an overwhelmingnumber of mutations have been uncovered in LMNA gene which leads to a plethora ofdiseasescollectivelytermedaslaminopathies.Laminopathicnucleiinthepatientsarecharacterizedbytheirmisshapennatureandfragilityduetoimproperlaminaformationunderneath the nuclear envelope. Almost 450 mutations of lamin A have been reported inpatients worldwide. This article uncovers the structural differences of Rod 2B R377L &amp;R377C with respect tothe wild type.

A Family with a Single LMNA Mutation Illustrates Diversity in Cardiac Phenotypes Associated with Laminopathic Progeroid Syndromes

The likely pathogenic variant c.407A&gt;T p.Asp136Val of the LMNA gene has been recently described in a young woman presenting with atypical progeroid syndrome, associated with severe aortic valve stenosis. We further describe the cardiovascular involvement associated with the syndrome in her family. We identified seven members with a general presentation suggestive of progeroid syndrome. All of them presented heart conduction abnormalities: degenerative cardiac diseases such as coronary artery disease (two subjects) and aortic stenosis (three subjects) occurred in the 3rd–5th decade, and a young patient developed a severe dilated cardiomyopathy, leading to death at 15 years of age. The likely pathogenic variant was found in all the patients who consented to carry out the genetic test. This diverse family cardiologic phenotype emphasizes the complex molecular background at play in lamin-involved cardiac diseases, and the need for early and thorough cardiac evaluations in patients with laminopathic progeroid syndromes.

Knockdown of LMNA inhibits Akt/β-catenin-mediated cell invasion and migration in clear cell renal cell carcinoma cells

ABSTRACT The LMNA gene encoding lamin A/C is amplified in some clear cell renal cell carcinoma (ccRCC) samples. Our data showed that depletion of the tumor suppressor PBRM1 can upregulate lamin A/C levels, and lamin A/C could interact with PBRM1. However, the role of lamin A/C in ccRCC is not yet fully understood. Our functional assays showed that although the proliferation ability was slightly impaired after LMNA depletion, the migration and invasion of ccRCC cells were significantly inhibited. This suppression was accompanied by a reduction in MMP2, MMP9, AKT/p-AKT, and Wnt/β-catenin protein levels. Our data therefore suggest that lamin A/C, as an interaction partner of the tumor suppressor PBRM1, plays a crucial role in tumor invasion and metastasis in ccRCC.

Effect of morphological change on the maturation of human induced pluripotent stem cell-derived cardiac tissue in rotating flow culture

IntroductionUnderstanding the critical factors for the maturation of human induced pluripotent stem cell (hiPSC)-derived cardiac tissue is important for further development of culture techniques. Rotating flow culture, where the tissues float in the culture medium by balancing its gravitational settling and the medium flow generated in rotating disk-shaped culture vessels, is one of culture systems used for tissue engineering. It has previously been demonstrated that rotating flow culture leads to the formation of matured cardiac tissue with higher levels of function and structure than the other culture systems. However, the detailed mechanisms underlying the maturation of cardiac tissue remain unclear. This study investigated the maturation process of hiPSC-derived cardiac tissue in rotating flow culture with a focus on morphological changes in the tissue, which is a trigger for maturation. MethodsThe cardiac tissue, which consisted of cardiomyocytes derived from hiPSCs, was cultured on the 3D scaffold of poly (lactic-co-glycolic) acid (PLGA)-aligned nanofibers, in rotating flow culture for 5 days. During the culture, the time profile of projected area of tissue and formation of maturation marker proteins (β-myosin heavy chain and Connexin-43), tissue structure, and formation of nuclear lamina proteins (Lamin A/C) were compared with that in static suspension culture. ResultsThe ratio of the projected area of tissue significantly decreased from Day 0 to Day 3 due to tissue shrinkage. In contrast, Western blot analysis revealed that maturation protein markers of cardiomyocytes significantly increased after Day 3. In addition, in rotating flow culture, flat-shaped nuclei and fiber-like cytoskeletal structures were distributed in the surface region of tissue where medium flow was continuously applied. Moreover, Lamin A/C, which are generally formed in differentiated cells owing to mechanical force across the cytoskeleton and critically affect the maturation of cardiomyocytes, were significantly formed in the tissue of rotating flow culture. ConclusionsIn this study, we found that spatial heterogeneity of tissue structure and tissue shrinkage occurred in rotating flow culture, which was not observed in static suspension culture. Moreover, from the quantitative analysis, it was also suggested that tissue shrinkage in rotating flow culture contributed its following tissue maturation. These findings showed one of the important characteristics of rotating flow culture which was not revealed in previous studies.

Progerin, an Aberrant Spliced Form of Lamin A, Is a Potential Therapeutic Target for HGPS.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder caused by the mutant protein progerin, which is expressed by the abnormal splicing of the LMNA gene. HGPS affects systemic levels, with the exception of cognition or brain development, in children, showing that cellular aging can occur in the short term. Studying progeria could be useful in unraveling the causes of human aging (as well as fatal age-related disorders). Elucidating the clear cause of HGPS or the development of a therapeutic medicine could improve the quality of life and extend the survival of patients. This review aimed to (i) briefly describe how progerin was discovered as the causative agent of HGPS, (ii) elucidate the puzzling observation of the absence of primary neurological disease in HGPS, (iii) present several studies showing the deleterious effects of progerin and the beneficial effects of its inhibition, and (iv) summarize research to develop a therapy for HGPS and introduce clinical trials for its treatment.

Abstract 084: Smooth Muscle Cullin-3 Regulates The Renal Baroreceptor Mechanism Via Lamin AC

Cullin-3 (CUL3) is a critical component of the CUL3-Ring-Ligase (CRL) ubiquitin ligase complex. Patients carrying mutations in CUL3 exhibit early onset of hypertension (HTN). We demonstrated that conditional ablation of CUL3 in vascular smooth muscle cells (VSMCs) results in vascular dysfunction, arterial stiffness, and severe HTN in mice. We hypothesized that 1) conditional deletion of CUL3 in VSMCs (S-CUL3KO) results in severe HTN via the renin-angiotensin system (RAS)-dependent mechanism, and 2) S-CUL3KO mice exhibit an impaired baroreceptor mechanism regulating renin. Mice carrying a conditional allele of CUL3 were bred with mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). S-CUL3KO mice exhibited elevated systolic blood pressure (SBP) 3 weeks after tamoxifen administration (S-CUL3KO: 164±1 vs. ISM-CRE: 124±4 mmHg, p&lt;0.05). To test the first hypothesis captopril or candesartan (10 mg/kg/day and 100 mg/kg/day in drinking water, respectively) were administered to S-CUL3KO once severe HTN was already established. Captopril or candesartan treatment for 1 week reversed elevated SBP to 117±4 mmHg (p&lt;0.05) and 127±2 mmHg (p&lt;0.05), respectively, in S-CUL3KO. Despite the severity of HTN in S-CUL3KO, the level of renin mRNA in the kidney was surprisingly unaltered (p&gt;0.05) when compared to the normotensive control mice. The protein expression of lamin AC, a crucial component of the nuclear mechanotransducer controlling renin expression, was significantly increased in S-CUL3KO kidneys (0.11±0.02 vs. 0.05±0.01 normalized RFU, p&lt;0.05). This suggests lamin AC may be a target protein of CUL3-ubiquitin ligase complex. Supporting this concept, treatment of A10 smooth muscle cells with MLN4914, an inhibitor of the Cullin pathway, results in a 2-fold increase in lamin AC expression (0.007±0.001 vs. 0.0154±0.0004, normalized RFU, p&lt;0.05). Furthermore, co-immunoprecipitation of CUL3 in HEK293 cells immunoprecipitated lamin AC. Our findings support the hypothesis that 1) CUL3 regulates RAS via the renal baroreceptor mechanism and 2) lamin AC is a potential target of the CRL3-mediated regulation. Understanding CUL3 target proteins might help the development of new strategies to mitigate HTN.

Functional Interactions of BAF and LEM Proteins in the Formation of Germ Cells

Recovery of the nuclear structure after cell division requires special interactions between the integral proteins of the inner nuclear membrane having a special LEM domain (LEMD), nuclear lamina proteins (lamins) and the conserved BAF protein that serves as a central link in these interactions, providing topological relationships between chromatin and nuclear envelope. The dynamic transformations of these protein ensembles in the mitotic cycle are characterized in detail at the molecular level, however, less attention is paid to the developing germ cells undergoing meiotic divisions, despite of their nuclei, especially in diplotene oocytes, differ significantly in structure from the somatic nucleus. This review summarizes the still relatively scarce experimental data proving the significance of functional interactions between BAF and LEMD proteins for gamete formation, from the selection of germline cells to the transformation of haploid spermatids into morphologically mature spermatozoa.

Abstract 124: Renin Cell Identity And Blood Pressure Homeostasis: Exploring The Role Of Endothelial Cell Cross-talk

Our previous studies using an Aortic Coarctation (AoCo) mouse model identified that renin-expressing juxta-glomerular (JG) cells respond to changes in perfusion pressure and Lamin A/C as a component of the renin cell baroreceptor. However, as the mechanisms for transmitting the extracellular pressure to the JG cells are unclear, we hypothesized that the endothelial cells (ECs) act as transducers of signals to the JG cells. Therefore in the current study, we aim 1) to identify the concurrent transcriptomic and epigenomic changes in JG cells and ECs in response to the changes in the perfusion pressure by single-nuclei multiomics approach 2) to define the contribution of EC-specific Lamin A/C (Lmna) in the EC-JG-cells cross-talk. We performed the AoCo surgeries on 3 months (M) old VE-Cad-Cre;Foxd1-Cre;mTmG mice where GFP is expressed simultaneously in Foxd1 and EC-lineage cells. We compared the single-cell gene expression and chromatin accessibility between the left (LK) and the right kidneys (RK) at 72h post-surgeries. Our study revealed: 1) JG cells increased significantly in the LK along with renin gene expression 2) ECs significantly increased by 10.73% in LK with a higher expression for genes promoting EC proliferation, morphogenesis, and immune response 3) JGs of the LK showed enrichment for the motifs Mbd1, Smad1, Smad5 indicating epigenetic changes promoting transcriptional regulation and TgfB1 signaling. Enrichment of Mybl1, Myb, and E2f2 motifs in the ECs of the LK suggests positive regulation of EC proliferation in response to low blood pressure (BP). Furthermore, deletion of the Lmna gene in the EC-lineage affected the BP responses in 4M old Lmna fl/fl ;VE-Cad.Cre mice. The difference between the carotid and femoral mean BP (MBP) measurements was more prominent in the wild type (WT) compared to the mutants at 72h post-AoCo [WT (n=7), Carotid MBP: 69.16±2.24 Femoral MBP: 63.28±2.33, P&lt;0.00001 Mutants (n=4); Carotid MBP: 75.98±3.06 Femoral MBP: 69.75±3.10, P&lt;0.05)]. Our current study shows that ECs undergo transcriptional and epigenetic alterations in response to changes in perfusion pressure. Deletion of Lmna in ECs reduces the differences in the BP between LK and RK, suggesting that it is important for sensing the changes in extracellular pressure.

Caenorhabditis elegans models for striated muscle disorders caused by missense variants of human LMNA.

Striated muscle laminopathies caused by missense mutations in the nuclear lamin gene LMNA are characterized by cardiac dysfunction and often skeletal muscle defects. Attempts to predict which LMNA variants are pathogenic and to understand their physiological effects lag behind variant discovery. We created Caenorhabditis elegans models for striated muscle laminopathies by introducing pathogenic human LMNA variants and variants of unknown significance at conserved residues within the lmn-1 gene. Severe missense variants reduced fertility and/or motility in C. elegans. Nuclear morphology defects were evident in the hypodermal nuclei of many lamin variant strains, indicating a loss of nuclear envelope integrity. Phenotypic severity varied within the two classes of missense mutations involved in striated muscle disease, but overall, variants associated with both skeletal and cardiac muscle defects in humans lead to more severe phenotypes in our model than variants predicted to disrupt cardiac function alone. We also identified a separation of function allele, lmn-1(R204W), that exhibited normal viability and swimming behavior but had a severe nuclear migration defect. Thus, we established C. elegans avatars for striated muscle laminopathies and identified LMNA variants that offer insight into lamin mechanisms during normal development.

OPA1, a molecular regulator of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a disease with no specific treatment, poor prognosis and high mortality. During DCM development, there is apoptosis, mitochondrial dynamics imbalance and changes in cristae structure. Optic atrophy 1 (OPA1) appears at high frequency in these three aspects. DCM LMNA (LaminA/C) gene mutation can activate TP53, and the study of P53 shows that P53 affects OPA1 through Bak/Bax and OMA1 (a metalloprotease). OPA1 can be considered the missing link between DCMp53 and DCM apoptosis, mitochondrial dynamics imbalance and changes in cristae structure. OPA1 regulates apoptosis by regulating the release of cytochrome c from the mitochondrial matrix through CJs (crisp linkages, located in the inner mitochondrial membrane) and unbalances mitochondrial fusion and fission by affecting mitochondrial inner membrane (IM) fusion. OPA1 is also associated with the formation and maintenance of mitochondrial cristae. OPA1 is not the root cause of DCM, but it is an essential mediator in P53 mediating the occurrence and development of DCM, so OPA1 also becomes a molecular regulator of DCM. This review discusses the implication of OPA1 for DCM from three aspects: apoptosis, mitochondrial dynamics and ridge structure.

Nuclear damage in LMNA mutant iPSC-derived cardiomyocytes is associated with impaired lamin localization to the nuclear envelope.

The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which comprise a major part of the nuclear lamina, provide mechanical support to the nucleus, and participate in diverse intracellular signaling. LMNA mutations give rise to a collection of diseases called laminopathies, including dilated cardiomyopathy (LMNA-DCM) and muscular dystrophies. Although nuclear deformities are a hallmark of LMNA-DCM, the role of nuclear abnormalities in the pathogenesis of LMNA-DCM remains incompletely understood. Using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA mutant patients and healthy controls, we show that LMNA mutant iPSC-CM nuclei have altered shape or increased size compared to healthy control iPSC-CM nuclei. The LMNA mutation exhibiting the most severe nuclear deformities, R249Q, additionally caused reduced nuclear stiffness and increased nuclear fragility. Importantly, for all cell lines, the degree of nuclear abnormalities corresponded to the degree of Lamin A/C and Lamin B1 mislocalization from the nuclear envelope. The mislocalization was likely due to altered assembly of Lamin A/C. Collectively, these results point to the importance of correct lamin assembly at the nuclear envelope in providing mechanical stability to the nucleus and suggest that defects in nuclear lamina organization may contribute to the nuclear and cellular dysfunction in LMNA-DCM.

Hutchinson-Gilford progeria patient-derived cardiomyocyte model of carrying LMNA gene variant c.1824 C > T.

Cardiovascular diseases, atherosclerosis, and strokes are the most common causes of death in patients with Hutchinson-Gilford progeria syndrome (HGPS). The LMNA variant c.1824C > T accounts for ~ 90% of HGPS cases. The detailed molecular mechanisms of Lamin A in the heart remain elusive due to the lack of appropriate in vitro models. We hypothesize that HGPS patient's induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMCs) will provide a model platform to study the cardio-pathologic mechanisms associated with HGPS. To elucidate the effects of progerin in cardiomyocytes, we first obtained skin fibroblasts (SFs) from a de-identified HGPS patient (hPGP1, proband) and both parents from the Progeria Research Foundation. Through Sanger sequencing and restriction fragment length polymorphism, with the enzyme EciI, targeting Lamin A, we characterized hPGP1-SFs as heterozygous mutants for the LMNA variant c.1824 C > T. Additionally, we performed LMNA exon 11 bisulfite sequencing to analyze the methylation status of the progeria cells. Furthermore, we reprogrammed the three SFs into iPSCs and differentiated them into iCMCs, which gained a beating on day 7. Through particle image velocimetry analysis, we found that hPGP1-iCMCs had an irregular contractile function and decreased cardiac-specific gene and protein expressions by qRT-PCR and Western blot. Our progeria-patient-derived iCMCs were found to be functionally and structurally defective when compared to normal iCMCs. This in vitro model will help in elucidating the role of Lamin A in cardiac diseases and the cardio-pathologic mechanisms associated with progeria. It provides a new platform for researchers to study novel treatment approaches for progeria-associated cardiac diseases.

Abstract P3134: Impaired Calcium Homeostasis Underlies Lamin A/C Mutation R331Q Associated Atrial Fibrillation

Background: Atrial fibrillation (AF) is one of the first cardiac manifestations in patients harboring mutations in nucleoskeletal protein lamin A/C (LMNA). However, the underlying arrhythmogenic mechanisms are unclear. Objective: We hypothesize that impaired intracellular Ca 2+ -homeostasis contribute to LMNA mutation-associated atrial arrhythmogenesis. Methods: LMNA mutation R331Q was introduced in a healthy control induced pluripotent stem cell (iPSC) line using CRISPR/Cas9 and cells were differentiated into atrial iPSC-derived cardiomyocytes (iPSC-CMs). L-type Ca 2+ current and intracellular Ca 2+ concentration were measured using patch-clamp technique and epifluorescence microscopy (Fluo-3), respectively. Results: R331Q iPSC-CMs demonstrated increased L-type Ca 2+ current density followed by increased amplitude of systolic Ca 2+ transient (CaT) compared to isogenic control (Fig. A). Sarcoplasmic reticulum (SR) Ca 2+ content was evaluated from caffeine-induced CaT amplitude and integrated Na + -Ca 2+ exchange current, and found to be greater in R331Q iPSC-CMs (Fig. B). Confocal line scan analysis revealed an increased Ca 2+ spark frequency in R331Q iPSC-CMs (Fig. C). In addition, by applying a stepwise increase in stimulation frequency (1-5 Hz), R331Q iPSC-CMs developed Ca 2+ alternans at lower threshold frequency (Fig. D). Conclusions: LMNA mutation R331Q is associated with disturbed intracellular Ca 2+ cycling, promoting occurrence of diastolic Ca 2+ sparks and arrhythmogenic Ca 2+ alternans. Collectively, our findings suggest that impaired Ca 2+ homeostasis may contribute to atrial arrhythmogenesis in patients harboring LMNA mutations.

The stability of the myelinating oligodendrocyte transcriptome is regulated by the nuclear lamina

Oligodendrocytes are specialized cells that insulate and support axons with their myelin membrane, allowing proper brain function. Here, we identify lamin A/C (LMNA/C) as essential for transcriptional and functional stability of myelinating oligodendrocytes. We show that LMNA/C levels increase with differentiation of progenitors and that loss of Lmna in differentiated oligodendrocytes profoundly alters their chromatin accessibility and transcriptional signature. Lmna deletion in myelinating glia is compatible with normal developmental myelination. However, altered chromatin accessibility is detected in fully differentiated oligodendrocytes together with increased expression of progenitor genes and decreased levels of lipid-related transcription factors and inner mitochondrial membrane transcripts. These changes are accompanied by altered brain metabolism, lower levels of myelin-related lipids, and altered mitochondrial structure in oligodendrocytes, thereby resulting in myelin thinning and the development of a progressively worsening motor phenotype. Overall, our data identify LMNA/C as essential for maintaining the transcriptional and functional stability of myelinating oligodendrocytes.

The nuclear lamina is required for proper development and nuclear shape distortion in tomato.

The nuclear lamina in plant cells is composed of plant-specific proteins, including Nuclear Matrix Constituent Proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and helps stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared to cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared to Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.

Nuclear proteostasis imbalance in laminopathy-associated premature aging diseases.

Laminopathies are a group of rare genetic disorders with heterogeneous clinical phenotypes such as premature aging, cardiomyopathy, lipodystrophy, muscular dystrophy, microcephaly, epilepsy, and so on. The cellular phenomena associated with laminopathy invariably show disruption of nucleoskeleton of lamina due to deregulated expression, localization, function, and interaction of mutant lamin proteins. Impaired spatial and temporal tethering of lamin proteins to the lamina or nucleoplasmic aggregation of lamins are the primary molecular events that can trigger nuclear proteotoxicity by modulating differential protein-protein interactions, sequestering quality control proteins, and initiating a cascade of abnormal post-translational modifications. Clearly, laminopathic cells exhibit moderate to high nuclear proteotoxicity, raising the question of whether an imbalance in nuclear proteostasis is involved in laminopathic diseases, particularly in diseases of early aging such as HGPS and laminopathy-associated premature aging. Here, we review nuclear proteostasis and its deregulation in the context of lamin proteins and laminopathies.

Nanotube patterning reduces macrophage inflammatory response via nuclear mechanotransduction

The inflammatory immune environment surrounding titanium bone implants determines the formation of osseointegration, and nanopatterning on implant surfaces modulates the immune microenvironment in the implant region. Among many related mechanisms, the mechanism by which nanopatterning controls macrophage inflammatory response still needs to be elucidated. In this paper, we found that inhibition of the nuclear envelope protein lamin A/C by titania nanotubes (TNTs) reduced the macrophage inflammatory response. Knockdown of lamin A/C reduced macrophage inflammatory marker expression, while overexpression of lamin A/C significantly elevated inflammatory marker expression. We further found that suppression of lamin A/C by TNTs limited actin polymerization, thereby reducing the nuclear translocation of the actin-dependent transcriptional cofactor MRTF-A, which subsequently reduced the inflammatory response. In addition, emerin, which is a key link between lamin A/C and actin, was delocalized from the nucleus in response to mechanical stimulation by TNTs, resulting in reduced actin organization. Under inflammatory conditions, TNTs exerted favourable osteoimmunomodulatory effects on the osteogenic differentiation of mouse bone marrow-derived stem cells (mBMSCs) in vitro and osseointegration in vivo. This study shows and confirms for the first time that lamin A/C-mediated nuclear mechanotransduction controls macrophage inflammatory response, and this study provides a theoretical basis for the future design of immunomodulatory nanomorphologies on the surface of metallic bone implants.