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LMNA-related dilated cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C (LMNA) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. The molecular mechanisms of the disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA-related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA-mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (four from Patients and eight from Controls) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for cardiac progenitors to cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Data integration and comparative analyses of Patient and Control cells found cell type and lineage-specific differentially expressed genes (DEGs) with enrichment, supporting pathway dysregulation. Top DEGs and enriched pathways included 10 ZNF genes and RNA polymerase II transcription in pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CMs; LMNA and epigenetic regulation, as well as DDIT4 and mTORC1 signaling in EPDCs. Top DEGs also included XIST and other X-linked genes, six imprinted genes (SNRPN, PWAR6, NDN, PEG10, MEG3, MEG8), and enriched gene sets related to metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs, as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.
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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 -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 with 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 suggests that defects in nuclear lamina organization may contribute to the nuclear and cellular dysfunction in LMNA-DCM.
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- Award ID(s):
- 2022048
- PAR ID:
- 10523259
- Editor(s):
- Discher, Dennis
- Publisher / Repository:
- American Society of Cell Biology
- Date Published:
- Journal Name:
- Molecular Biology of the Cell
- Volume:
- 34
- Issue:
- 12
- ISSN:
- 1059-1524
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract 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.more » « less
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ABSTRACT Lamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is crucial for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna–/–) MEFs, and all LaA constructs prevented increased nuclear envelope ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit emerin to the nuclear membrane in Lmna–/– MEFs. Our finding that tags impede some LaA functions but not others might explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.more » « less
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Abstract Lamins A and C, encoded by theLMNAgene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation.LMNAmutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which manyLMNAmutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragileLmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts ofLmnamutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity inLmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.more » « less
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Cell migration through narrow spaces is essential in wound healing and metastatic spread of cancer. Cells must deform the large nucleus to fit through constricting channels. To understand the role of the nuclear lamina in limiting cell migration through constrictions, we imaged it in cells migrating through periodic constricting channels in a microdevice. The lamina underwent cycles of wrinkling and smoothing as the nucleus changed from an irregular, rounded shape in the wide channel regions between constrictions to a smooth, hourglass shape as the nucleus passed through the center of a constriction. The laminar surface area of nuclei within constrictions was measured to be at or above the computationally predicted threshold area for the nuclear volume. The channels excluded control nuclei that had insufficient excess surface area, but not nuclei lacking lamin A/C. Thus, the excess surface area of the nuclear lamina enables cell migration through constricting channels.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1091/mbc.E21-10-0527
