Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes

•Pathogenic LMNA variants disrupt peripheral chromatin in a cell-type-specific manner•Loss of lamina-bound chromatin occurs in regions with specific characteristics•Peripheral chromatin organization maintains silencing of alternative fate genes Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types. Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types. Genome-lamina interactions provide a critical layer of gene regulation, but it remains unclear how these interactions affect cellular identity and disease. The nuclear lamina is a filamentous network of LAMIN A/C (LMNA), LAMIN B1 (LB1), and LAMIN B2 proteins on the inner nuclear surface. LMNA mutations result in gross nuclear abnormalities (Burke and Stewart, 2006Burke B. Stewart C.L. The laminopathies: the functional architecture of the nucleus and its contribution to disease.Annu. Rev. Genomics Hum. Genet. 2006; 7: 369-405Crossref PubMed Scopus (127) Google Scholar; Worman and Bonne, 2007Worman H.J. Bonne G. “Laminopathies”: a wide spectrum of human diseases.Exp. Cell Res. 2007; 313: 2121-2133Crossref PubMed Scopus (460) Google Scholar) and are the second most common cause of familial dilated cardiomyopathy (DCM) (Taylor et al., 2003Taylor M.R. Fain P.R. Sinagra G. Robinson M.L. Robertson A.D. Carniel E. Di Lenarda A. Bohlmeyer T.J. Ferguson D.A. Brodsky G.L. et al.Familial Dilated Cardiomyopathy Registry Research GroupNatural history of dilated cardiomyopathy due to lamin A/C gene mutations.J. Am. Coll. Cardiol. 2003; 41: 771-780Crossref PubMed Scopus (335) Google Scholar). There is a paucity of data reconciling how germline LMNA mutations result in tissue-specific phenotypes. LMNA mutants can alter normal nuclear rigidity (Houben et al., 2007Houben F. Ramaekers F.C. Snoeckx L.H. Broers J.L. Role of nuclear lamina-cytoskeleton interactions in the maintenance of cellular strength.Biochim. Biophys. Acta. 2007; 1773: 675-686Crossref PubMed Scopus (97) Google Scholar; Lee et al., 2007Lee J.S. Hale C.M. Panorchan P. Khatau S.B. George J.P. Tseng Y. Stewart C.L. Hodzic D. Wirtz D. Nuclear lamin A/C deficiency induces defects in cell mechanics, polarization, and migration.Biophys. J. 2007; 93: 2542-2552Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), but most tissues express LMNA, and soft and stiff tissues are affected by LMNA mutations (Burke and Stewart, 2006Burke B. Stewart C.L. The laminopathies: the functional architecture of the nucleus and its contribution to disease.Annu. Rev. Genomics Hum. Genet. 2006; 7: 369-405Crossref PubMed Scopus (127) Google Scholar). LMNA mutants are implicated in altered gene expression via aberrations in signal transduction or genome organization (Hutchison, 2002Hutchison C.J. Lamins: building blocks or regulators of gene expression?.Nat. Rev. Mol. Cell Biol. 2002; 3: 848-858Crossref PubMed Scopus (245) Google Scholar; Worman, 2018Worman H.J. Cell signaling abnormalities in cardiomyopathy caused by lamin A/C gene mutations.Biochem. Soc. Trans. 2018; 46: 37-42Crossref PubMed Scopus (14) Google Scholar; Wu et al., 2011Wu W. Muchir A. Shan J. Bonne G. Worman H.J. Mitogen-activated protein kinase inhibitors improve heart function and prevent fibrosis in cardiomyopathy caused by mutation in lamin A/C gene.Circulation. 2011; 123: 53-61Crossref PubMed Scopus (120) Google Scholar). The nuclear lamina associates with large chromatin regions called LAMIN B1-associated domains (LADs) (Guelen et al., 2008Guelen L. Pagie L. Brasset E. Meuleman W. Faza M.B. Talhout W. Eussen B.H. de Klein A. Wessels L. de Laat W. van Steensel B. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1249) Google Scholar). Genes in LADs are transcriptionally repressed, and some LADs are repositioned away from or to the lamina during differentiation in a cell-type-specific manner (Meister et al., 2010Meister P. Towbin B.D. Pike B.L. Ponti A. Gasser S.M. The spatial dynamics of tissue-specific promoters during C. elegans development.Genes Dev. 2010; 24: 766-782Crossref PubMed Scopus (157) Google Scholar; Peric-Hupkes et al., 2010Peric-Hupkes D. Meuleman W. Pagie L. Bruggeman S.W. Solovei I. Brugman W. Gräf S. Flicek P. Kerkhoven R.M. van Lohuizen M. et al.Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.Mol. Cell. 2010; 38: 603-613Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). In worms, perinuclear chromatin sequestration contributes to lineage restriction by stabilizing cell fate commitment (Gonzalez-Sandoval et al., 2015Gonzalez-Sandoval A. Towbin B.D. Kalck V. Cabianca D.S. Gaidatzis D. Hauer M.H. Geng L. Wang L. Yang T. Wang X. et al.Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos.Cell. 2015; 163: 1333-1347Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). LAD positioning regulates mammalian organogenesis (Peric-Hupkes et al., 2010Peric-Hupkes D. Meuleman W. Pagie L. Bruggeman S.W. Solovei I. Brugman W. Gräf S. Flicek P. Kerkhoven R.M. van Lohuizen M. et al.Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.Mol. Cell. 2010; 38: 603-613Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar; Poleshko et al., 2017Poleshko A. Shah P.P. Gupta M. Babu A. Morley M.P. Manderfield L.J. Ifkovits J.L. Calderon D. Aghajanian H. Sierra-Pagán J.E. et al.Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.Cell. 2017; 171: 573-587.e14Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar; Robson et al., 2016Robson M.I. de Las Heras J.I. Czapiewski R. Lê Thành P. Booth D.G. Kelly D.A. Webb S. Kerr A.R.W. Schirmer E.C. Tissue-Specific Gene Repositioning by Muscle Nuclear Membrane Proteins Enhances Repression of Critical Developmental Genes during Myogenesis.Mol. Cell. 2016; 62: 834-847Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), but it is unclear whether compromised LAD organization impairs cell-type-specific gene expression. Pathogenic LMNA variants provide an attractive approach to unveil principles underlying the consequences of lamina-genome interactions. To this end, a C. elegans mutant lamin muscular dystrophy model displays muscle phenotypes and is rescued by restoring peripheral chromatin organization via loss of a peripheral chromatin tether (Harr et al., 2020Harr J.C. Schmid C.D. Muñoz-Jiménez C. Romero-Bueno R. Kalck V. Gonzalez-Sandoval A. Hauer M.H. Padeken J. Askjaer P. Mattout A. Gasser S.M. Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.Genes Dev. 2020; 34: 560-579Crossref PubMed Scopus (16) Google Scholar). Because a subset of LADs are cell type specific, mechanisms relevant to tissue-specific disease phenotypes are likely to be influenced by cellular context. This limits the interpretation of studies overexpressing variants or using immortalized cell lines (Mewborn et al., 2010Mewborn S.K. Puckelwartz M.J. Abuisneineh F. Fahrenbach J.P. Zhang Y. MacLeod H. Dellefave L. Pytel P. Selig S. Labno C.M. et al.Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation.PLoS ONE. 2010; 5: e14342Crossref PubMed Scopus (85) Google Scholar; Perovanovic et al., 2016Perovanovic J. Dell’Orso S. Gnochi V.F. Jaiswal J.K. Sartorelli V. Vigouroux C. Mamchaoui K. Mouly V. Bonne G. Hoffman E.P. Laminopathies disrupt epigenomic developmental programs and cell fate.Sci. Transl. Med. 2016; 8: 335ra58Crossref PubMed Scopus (60) Google Scholar). Also, only a subset of LADs reposition away from the lamina during differentiation, and often only a portion of a LAD is repositioned (Briand and Collas, 2020Briand N. Collas P. Lamina-associated domains: peripheral matters and internal affairs.Genome Biol. 2020; 21: 85Crossref PubMed Scopus (42) Google Scholar). Genomic regions have varying probabilities of re-localization to or from the lamina (Kind et al., 2013Kind J. Pagie L. Ortabozkoyun H. Boyle S. de Vries S.S. Janssen H. Amendola M. Nolen L.D. Bickmore W.A. van Steensel B. Single-cell dynamics of genome-nuclear lamina interactions.Cell. 2013; 153: 178-192Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, Kind et al., 2015Kind J. Pagie L. de Vries S.S. Nahidiazar L. Dey S.S. Bienko M. Zhan Y. Lajoie B. de Graaf C.A. Amendola M. et al.Genome-wide maps of nuclear lamina interactions in single human cells.Cell. 2015; 163: 134-147Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Thus, different types of peripheral chromatin may exist at the nuclear lamina with distinct mechanisms of establishment or maintenance, function, or genomic features. It is of great interest to understand whether peripheral chromatin is affected uniformly or stochastically by LMNA variants. Distinguishing LADs vulnerable to disruption from those that are resistant is vital to understand the mechanism of peripheral chromatin organization and the role of LADs in cellular identity and disease progression. We introduced a point mutation into one LMNA allele in a human induced pluripotent stem cell (hiPSC) line, resulting in a heterozygous T10I LAMIN A mutant, modeled after an individual with laminopathy. Mutant hiPSC-derived cardiomyocytes (hiPSC-CMs) demonstrated impaired physiology, dysmorphic nuclei, and disruption of a specific subset of peripheral chromatin regions characterized by greater gene density, higher expression, and lower LB1 enrichment. hiPSC-CMs carrying a pathogenic LMNA R541C mutation showed similar changes. These disruptions were specific to hiPSC-CMs; T10I and R541C hiPSC hepatocytes or hiPSC adipocytes did not demonstrate dysmorphic nuclei or LAD changes. Disrupted hiPSC-CM peripheral chromatin regions were enriched for genes and regulatory regions relevant to non-myocyte cell types, resulting in aberrant expression of genes ordinarily restricted to non-myocyte lineages. These data reveal that a subset of lamina-bound chromatin with definable molecular characteristics is disrupted in disease and may contribute to tissue-specific phenotypes observed in individuals with laminopathy. Genetic testing of a 33-year-old female with congestive heart failure requiring cardiac transplantation identified a heterozygous LMNA T10I (hereafter called T10I) mutation (Figure 1A). The woman’s family history was consistent with familial DCM; her father had heart failure requiring cardiac transplantation, and several relatives suffered from heart failure or sudden cardiac death. Explanted myocardium from the proband T10I individual revealed CMs with significantly larger and dysmorphic nuclei compared with myocardium from non-failing hearts or individuals with idiopathic DCM (Figures 1B, 1C, and S1A). We introduced the T10I mutation via CRISPR-Cas9 into one LMNA allele in hiPSCs from an unrelated healthy individual (Figure S1B). hiPSCs that acquired no mutation served as controls. We isolated independent control and T10I clones; assessment of the top 10 Cas9 recognition sites did not identify off-target effects (Figure S1C). We proceeded with two validated clones per genotype and differentiated control and T10I hiPSCs into TNNT2+ CMs (Figure 1D). Flow cytometry across multiple biological replicates confirmed that more than 85% of control and T10I hiPSC-CMs expressed TNNT2 and MLC2v (Figures 1E and S1D; Lian et al., 2013Lian X. Zhang J. Azarin S.M. Zhu K. Hazeltine L.B. Bao X. Hsiao C. Kamp T.J. Palecek S.P. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions.Nat. Protoc. 2013; 8: 162-175Crossref PubMed Scopus (868) Google Scholar; Zhu et al., 2011Zhu W.Z. Van Biber B. Laflamme M.A. Methods for the derivation and use of cardiomyocytes from human pluripotent stem cells.Methods Mol. Biol. 2011; 767: 419-431Crossref PubMed Scopus (59) Google Scholar). RNA sequencing (RNA-seq) analysis (control, n = 4; T10I, n = 3) confirmed nearly equivalent expression of the mutant and wild-type LMNA alleles in T10I hiPSC-CMs (Figure S1E). Immunoblotting confirmed LAMIN A and LAMIN C protein in control and mutant hiPSC-CMs but not undifferentiated cells (noting a reduction in T10I hiPSC-CMs) (Figure S1F). Transfection of IMR90 human fibroblasts with Emerald-tagged LAMIN A or LAMIN A T10I also showed a reduced epitope-tagged LAMIN A signal for T10I compared with the control but equivalent Emerald expression (Figure S1G). Lamina filaments regulate nuclear and cytoskeletal stiffness (Cho et al., 2019Cho S. Vashisth M. Abbas A. Majkut S. Vogel K. Xia Y. Ivanovska I.L. Irianto J. Tewari M. Zhu K. et al.Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest.Dev. Cell. 2019; 49: 920-935.e5Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar; Lammerding et al., 2004Lammerding J. Schulze P.C. Takahashi T. Kozlov S. Sullivan T. Kamm R.D. Stewart C.L. Lee R.T. Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction.J. Clin. Invest. 2004; 113: 370-378Crossref PubMed Scopus (756) Google Scholar; McKee et al., 2011McKee C.T. Raghunathan V.K. Nealey P.F. Russell P. Murphy C.J. Topographic modulation of the orientation and shape of cell nuclei and their influence on the measured elastic modulus of epithelial cells.Biophys. J. 2011; 101: 2139-2146Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar; Swift et al., 2013Swift J. Ivanovska I.L. Buxboim A. Harada T. Dingal P.C. Pinter J. Pajerowski J.D. Spinler K.R. Shin J.W. Tewari M. et al.Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation.Science. 2013; 341: 1240104Crossref PubMed Scopus (1079) Google Scholar; Worman and Bonne, 2007Worman H.J. Bonne G. “Laminopathies”: a wide spectrum of human diseases.Exp. Cell Res. 2007; 313: 2121-2133Crossref PubMed Scopus (460) Google Scholar). We measured the elastic and viscoelastic properties of single hiPSC-CMs using atomic force microscopy (AFM; Figures 1F and 1G). T10I hiPSC-CMs had a decreased Young’s modulus at physiological rates of mechanical stimulation (2-Hz indentation at 25 V; Figure 1F), impaired elastic properties at low rates of stimulation, and loss of normal viscoelasticity at high rates of stimulation (Figure 1G), consistent with other laminopathy models (Hale et al., 2008Hale C.M. Shrestha A.L. Khatau S.B. Stewart-Hutchinson P.J. Hernandez L. Stewart C.L. Hodzic D. Wirtz D. Dysfunctional connections between the nucleus and the actin and microtubule networks in laminopathic models.Biophys. J. 2008; 95: 5462-5475Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar; Khatau et al., 2009Khatau S.B. Hale C.M. Stewart-Hutchinson P.J. Patel M.S. Stewart C.L. Searson P.C. Hodzic D. Wirtz D. A perinuclear actin cap regulates nuclear shape.Proc. Natl. Acad. Sci. USA. 2009; 106: 19017-19022Crossref PubMed Scopus (388) Google Scholar). A Fluo-4 fluorescence calcium reporter assay demonstrated an unchanged amplitude of spontaneous calcium transients in T10I hiPSC-CMs, but mutants showed a faster time to peak calcium concentration and lower basal fluorescence compared with control cells (Figure 1H). Multiple indices of myocyte contraction performed in single hiPSC-CMs or those grown in 3D micropatterned cultures were reduced in T10I compared with control cells (Figures 1I and 1J). These data indicate that T10I hiPSC-CMs recapitulate physiological abnormalities observed in individuals with LMNA mutations and DCM. Nuclear blebbing and rupture are abnormal phenotypes associated with laminopathies (Burke and Stewart, 2006Burke B. Stewart C.L. The laminopathies: the functional architecture of the nucleus and its contribution to disease.Annu. Rev. Genomics Hum. Genet. 2006; 7: 369-405Crossref PubMed Scopus (127) Google Scholar; de Leeuw et al., 2018de Leeuw R. Gruenbaum Y. Medalia O. Nuclear Lamins: Thin Filaments with Major Functions.Trends Cell Biol. 2018; 28: 34-45Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar; Vergnes et al., 2004Vergnes L. Péterfy M. Bergo M.O. Young S.G. Reue K. Lamin B1 is required for mouse development and nuclear integrity.Proc. Natl. Acad. Sci. USA. 2004; 101: 10428-10433Crossref PubMed Scopus (292) Google Scholar; Worman and Bonne, 2007Worman H.J. Bonne G. “Laminopathies”: a wide spectrum of human diseases.Exp. Cell Res. 2007; 313: 2121-2133Crossref PubMed Scopus (460) Google Scholar). We visualized control and T10I hiPSC-CMs by immunofluorescence and blindly scored nuclei as normal (elliptical), mild defect (slight invagination), or severe defect (multiple invaginations or nuclear rupture/micronuclei). Control hiPSC-CMs demonstrated mostly normal nuclei. T10I hiPSC-CMs showed significantly more mild and severely defective nuclei on days 25 (mid-point) and 45 (later time point) of CM differentiation (Figures 2A and 2B ), which was particularly noticeable in multinucleated cells. Severely defective nuclei were also observed in mutants on days 9–10, prior to onset of gross contraction (Figure S1H). Consistent with previous work (Kind et al., 2013Kind J. Pagie L. Ortabozkoyun H. Boyle S. de Vries S.S. Janssen H. Amendola M. Nolen L.D. Bickmore W.A. van Steensel B. Single-cell dynamics of genome-nuclear lamina interactions.Cell. 2013; 153: 178-192Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar; Poleshko et al., 2017Poleshko A. Shah P.P. Gupta M. Babu A. Morley M.P. Manderfield L.J. Ifkovits J.L. Calderon D. Aghajanian H. Sierra-Pagán J.E. et al.Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.Cell. 2017; 171: 573-587.e14Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar; See et al., 2019See K. Lan Y. Rhoades J. Jain R. Smith C.L. Epstein J.A. Lineage-specific reorganization of nuclear peripheral heterochromatin and H3K9me2 domains.Development. 2019; 146: dev174078Crossref PubMed Scopus (11) Google Scholar), histone H3 lysine 9 dimethylated (H3K9me2) chromatin was enriched at the lamina in control cells (Figures 2A–2C), whereas severely defective (Figure 2A) and mildly defective (Figure 2C) T10I hiPSC-CMs showed reduced H3K9me2 enrichment immediately adjacent to the nuclear lamina. We hypothesized that T10I disrupts peripheral chromatin organization in hiPSC-CMs. LB1 is exclusive to the periphery whereas LAMIN A/C is not (Gesson et al., 2016Gesson K. Rescheneder P. Skoruppa M.P. von Haeseler A. Dechat T. Foisner R. A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha.Genome Res. 2016; 26: 462-473Crossref PubMed Scopus (108) Google Scholar). LB1-occupied chromatin represents the majority of peripheral LAMIN A/C-bound chromatin (Discussion; Meuleman et al., 2013Meuleman W. Peric-Hupkes D. Kind J. Beaudry J.B. Pagie L. Kellis M. Reinders M. Wessels L. van Steensel B. Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence.Genome Res. 2013; 23: 270-280Crossref PubMed Scopus (259) Google Scholar). We confirmed LB1 antibody specificity for chromatin immunoprecipitation (ChIP; Figure S2A) and performed ChIP sequencing (ChIP-seq) on day 25 control and T10I hiPSC-CMs (Figure 2D; Table S1). We merged biological replicates with high reproducibility (control, n = 3; T10I, n = 4) (Figure S2E) and identified LAMIN B1-associated domains (LADs) using Enriched Domain Detector (EDD) (Figure 2D; Lund et al., 2014Lund E. Oldenburg A.R. Collas P. Enriched domain detector: a program for detection of wide genomic enrichment domains robust against local variations.Nucleic Acids Res. 2014; 42: e92Crossref PubMed Scopus (65) Google Scholar). In control and T10I hiPSC-CMs, we identified 750–800 LADs (Table S1) occupying ∼25% of the genome. Approximately 18.5% of LAD coverage was shared (shared LADs), with ∼6.7% of control LAD coverage lost in T10I cells (control-only LADs) and ∼6.0% of T10I LAD coverage gained and unique to T10I cells (T10I-only LADs) (Figure 2E; Table S1). Parallel trends were observed in day 45 control and T10I hiPSC-CM ChIP-seq (control and T10I, n = 4; Figures S2B and S2C; Table S1). RNA-seq on day 25 (Table S2) confirmed that genes in control and T10I hiPSC-CM LADs are significantly repressed relative to non-LAD genes (Figure 2F). To determine the consistency of pathologic LMNA variants, we established a second set of hiPSC lines harboring the mutation LMNA R541C from an individual (hereafter called R541C; Figure S2D). The R541C individual also presented with DCM (explanted myocardium was not available for further analysis). We validated independent clones via sequencing for off-target effects (Figure S2E) and proceeded with two validated clones. Flow cytometry indicated consistent and efficient differentiation (Figure S2F). RNA-seq on day 25 (control and R541C, n = 4; Table S2) confirmed equal transcription from mutant and wild-type LMNA alleles in the heterozygous mutants (Figure S2G), and LAMIN A and C proteins were confirmed by immunoblotting (Figure S2H). Validated control lines were generated during construction of mutants and confirmed to not harbor T10I/R541C mutations. We combined datasets from the control lines to create a union control for subsequent analyses. Similar to T10I, AFM of R541C hiPSC-CMs showed impaired elastic properties at physiologic rates of mechanical stimulation (2 Hz) and decreased viscoelastic response to a range of stimulations (Figures S3A and S3B). R541C hiPSC-CMs also demonstrated a significantly faster time to peak cytosolic calcium concentration compared with control cells and slightly lower basal calcium concentrations (Figure S3C). R541C hiPSC-CMs showed a significant subset of cells with mild and severe nuclear morphology defects and disruption of H3K9me2 at the nuclear periphery (Figure S3D). We defined LADs (Table S1) from LB1 ChIP-seq in R541C hiPSC-CMs (n = 3; Figure S3E) and confirmed that LAD genes were relatively less expressed compared with non-LAD genes (Figure S3F; Table S2). Principal-component analysis (PCA) of the hiPSC-CM ChIP-seq data showed distinct separation of T10I and R541C LADs from control LADs (Figure S3G). Domains of H3K9me2-modified chromatin (H3K9 dimethylated domains [KDDs]) closely mirror LADs under physiological conditions (Poleshko et al., 2017Poleshko A. Shah P.P. Gupta M. Babu A. Morley M.P. Manderfield L.J. Ifkovits J.L. Calderon D. Aghajanian H. Sierra-Pagán J.E. et al.Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.Cell. 2017; 171: 573-587.e14Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). We performed H3K9me2 ChIP-seq in control, T10I, and R541C hiPSC-CMs and defined KDDs (control and T10I, n = 4; R541C, n = 3; Figure S3A; Table S1). As with LADs, genes in KDDs were relatively less expressed compared with genes outside of KDDs (Figure S4B). PCA of KDDs and LADs from all hiPSC-CM datasets (Figure 2G) showed separation of mutant LADs and KDDs from controls. LADs from both mutants clustered together, suggesting a consistent LAD defect. PCA also showed greater separation between LB1 and H3K9me2 in mutant compared with control hiPSC-CMs, suggesting that LAD/KDD co-occupancy may be reduced in the mutants. Because individual replicate data were combined for genomic analyses (unless indicated otherwise), any biases arising from differentiation or ChIP variability are attenuated in the final data. Collectively, these data establish that two different LMNA variants result in grossly abnormal nuclei and disruption of chromatin-lamina interactions, as defined by LB1 or H3K9me2 occupancy in hiPSC-CMs. Laminopathy phenotypes are often tissue restricted. Thus, we assessed T10I and R541C in different cell types. We differentiated control, T10I, and R541C hiPSCs into hepatocytes (hiPSC-heps) (Cai et al., 2008Cai J. DeLaForest A. Fisher J. Urick A. Wagner T. Twaroski K. Cayo M. Nagaoka M. Duncan S.A. Protocol for directed differentiation of human pluripotent stem cells toward a hepatocyte fate.StemBook. 2008; https://doi.org/10.3824/stembook.1.52.1Crossref Google Scholar) and confirmed ALBUMIN and LAMIN A/C protein on day 23 (Figures S4C and S4D). Clinical laminopathy phenotypes have been observed in hepatocytes, but they are less prevalent than cardiac phenotypes (Brady et al., 2018Brady G.F. Kwan R. Bragazzi Cunha J. Elenbaas J.S. Omary M.B. Lamins and Lamin-Associated Proteins in Gastrointestinal Health and Disease.Gastroenterology. 2018; 154: 1602-1619.e1Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar; Rankin and Ellard, 2006Rankin J. Ellard S. The laminopathies: a clinical review.Clin. Genet. 2006; 70: 261-274Crossref PubMed Scopus (146) Google Scholar). The T10I individual presented with steatohepatitis with unclear etiology (Hussain et al., 2018Hussain I. Patni N. Ueda M. Sorkina E. Valerio C.M. Cochran E. Brown R.J. Peeden J. Tikhonovich Y. Tiulpakov A. et al.A Novel Generalized Lipodystrophy-Associated Progeroid Syndrome Due to Recurrent Heterozygous LMNA p.T10I Mutation.J. Clin. Endocrinol. Metab. 2018; 103: 1005-1014Crossref PubMed Scopus (30) Google Scholar), but control, T10I, and R541C hiPSC-heps showed no clear differences in nuclear morphology, H3K9me2 staining, or cellular stiffness (Figures 3A, 3B, S4E, and S4F). We performed LB1 and H3K9me2 ChIP-seq and defined LADs and KDDs in control, T10I, and R541C hiPSC-heps (each genotype, n = 2 replicates/ChIP condition; Figures 3C, 3D, and S4G; Table S1). PCA and visual inspection of the data showed close clustering of mutant LADs and KDDs with control hiPSC-hep LADs and KDDs unlike in hiPSC-CMs (Figures 3D and S4H). We also differentiated hiPSCs into adipocytes (hiPSC-adips; Su et al., 2018Su S. Guntur A.R. Nguyen D.C. Fakory S.S. Doucette C.C. Leech C. Lotana H. Kelley M. Kohli J. Martino J. et al.A Renewable Source of Human Beige Adipocytes for Development of Therapies to Treat Metabolic Syndrome.Cell Rep. 2018; 25: 3215-3228.e9Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and confirmed LAMIN A/C protein (Figure S4I) and comparable lipid accumulation by BODIPY staining (Merrick et al., 2019Merrick D. Sakers A. Irgebay Z. Okada C. Calvert C. Morley M.P. Percec I. Seale P. Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.Science. 2019; 364: eaav2501https://doi.org/10.1126/science.aav2501Crossref PubMed Scopus (174) Google Scholar) across all genotypes (Figure S4J). A portion of T10I hiPSC-adips had more mildly defective nuclei than control cells but few severely defective nuclei, unlike hiPSC-CMs (compare Figures 3E, S4J, and S5A with Figures 2A, 2B, and S3D). AFM showed reduced elasticity of T10I compared with control hiPSC-adips (Figure S4K), but the differences were minor compared with hiPSC-CMs (Figure S4L). hiPSC-adip LADs and KDDs (Table S1), defined from LB1 and H3K9me2 ChIP-seq (n = 2/genotype), also showed minor changes (Figures S5A and S5B; Table S1). LAD PCA (Figure 3F) or LAD/KDD PCA (Figure S5C) of all three cell types showed separation of mutant hiPSC-CM LADs or KDDs from controls, whereas mutant hiPSC-adips or hiPSC-heps showed minimal differences from cell-type controls (Figures 3F and S5C). Thus, T10I and R541C LADs and KDDs are preferentially disrupted in hiPSC-CMs. PCA also showed separation of control hiPSC-CMs,