Mutant lamins cause nuclear envelope rupture and DNA damage in skeletal muscle cells

Abstract

Mutations in the LMNA gene, which encodes the nuclear envelope (NE) proteins lamins A/C, cause Emery–Dreifuss muscular dystrophy, congenital muscular dystrophy and other diseases collectively known as laminopathies. The mechanisms responsible for these diseases remain incompletely understood. Using three mouse models of muscle laminopathies and muscle biopsies from individuals with LMNA-related muscular dystrophy, we found that Lmna mutations reduced nuclear stability and caused transient rupture of the NE in skeletal muscle cells, resulting in DNA damage, DNA damage response activation and reduced cell viability. NE and DNA damage resulted from nuclear migration during skeletal muscle maturation and correlated with disease severity in the mouse models. Reduction of cytoskeletal forces on the myonuclei prevented NE damage and rescued myofibre function and viability in Lmna mutant myofibres, indicating that myofibre dysfunction is the result of mechanically induced NE damage. Taken together, these findings implicate mechanically induced DNA damage as a pathogenic contributor to LMNA skeletal muscle diseases.

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Fig. 1: In vitro-differentiated primary myoblasts from Lmna KO, Lmna N195K and Lmna H222P mice recapitulate disease severity.
Fig. 2: Lmna mutant muscle cells have reduced nuclear stability.
Fig. 3: Lmna mutant myonuclei exhibit chromatin protrusions and NE ruptures.
Fig. 4: Lmna KO myonuclei show increased DNA damage in vitro and in vivo.
Fig. 5: Reducing forces on myonuclei prevents NE rupture and improves viability and contractility in Lmna KO myotubes.
Fig. 6: Human muscle biopsy tissues from individuals with LMNA muscular dystrophy show increased DNA damage.

Data availability

The data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data for Fig. 4c and Extended Data Fig. 8a are provided with the paper.

Code availability

MATLAB codes used for the micro-harpoon assay and micropipette aspiration analysis are available on request.

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Acknowledgements

The authors thank C. Stewart for providing the Lmna KO and Lmna N195K mouse models; E. Gomes for help with the in vitro myocyte differentiation protocol; A. Corbin for in vivo and in vitro protrusion and cGAS analysis and in vivo Hsp90 localization analysis; R. Mount for optimization of single-fibre isolation and subsequent analysis; D. Huang for quantification of Lmna N195K skeletal muscle fibre cross-sectional areas; F. Vermeylen, S. Parry and L. Johnson from the Cornell Statistical Consulting Unit, and K. Strednak and the Cornell Center for Animal Resources and Education for help in maintaining Lmna mutant mice. Clinical data from LMNA individuals were provided by K. D. Mathews (Vice Chair for Clinical Investigation, Director, Muscular Dystrophy Clinic, Director, Iowa Neuromuscular Program, Professor of Pediatrics–Neurology and Professor of Neurology). Technical assistance with human muscle immunohistochemistry was provided by N. M. Shaw and M. R. Ketterer (University of Iowa). This work was supported by awards from the National Institutes of Health (nos. R01 HL082792 and U54 CA210184 to J.L.); the Department of Defense Breast Cancer Research Program (Breakthrough Award, no. BC150580 to J.L.); the National Science Foundation (CAREER Award, nos. CBET-1254846 and MCB-1715606 to J.L.); the Muscular Dystrophy Association (Development Award, no. MDA603238 to T.J.K. and no. 477283 to L.L.W); a Fleming Postdoctoral Fellowship, to T.J.K; National Science Foundation Graduate Research Fellowships (nos. 2013160437 to A.J.E. and 2014163403 to G.R.F.); a Burroughs Welcome Fund Collaborative Research Travel Grant (no. 1017502, to L.L.W.); the University of Iowa Wellstone Muscular Dystrophy Cooperative Research Center (nos. U54 and NS053672, to S.A.M); and generous gifts from the Mills family to J.L. This work was performed in part at CNF, a member of the National Nanotechnology Coordinated Infrastructure, supported by the National Science Foundation (grant no. NNCI-1542081).

Author information

A.J.E., T.J.K., G.R.F., P.I., L.L.W. and J.L. contributed to the conception and design of the work. A.J.E., T.J.K., G.R.F., P.I., J.P., S.I. and S.A.M. contributed to data acquisition and analysis. A.J.E., T.J.K., G.R.F., L.L.W., G.B. and J.L. contributed to interpretation of data. A.J.E., T.J.K., G.R.F., L.L.W. and J.L contributed to drafting of the manuscript. All authors contributed to editing the manuscript.

Correspondence to Jan Lammerding.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Lmna KO and Lmna N195K have reduced contractility and experience nuclear loss.

a, Quantification of myofibre contraction at day 10 of differentiation. Fibres were assigned contraction scores from 0 (worst) to 4 (best) based on the percentage of cells that were visually contracting. Data points indicate results from n independent primary cell lines for each genotype. Significance determined by one-way ANOVA, using Benjamini and Yecutieli (low power) multiple comparisons. b, Quantification of the change in nuclear number between day 5 and day 10 of differentiation. Data points indicate results from n independent primary cell lines for each genotype. Significance determined by one-way ANOVA, using Tukey’s correction for multiple comparisons. All bar plots show mean value ± standard error of the mean.

Extended Data Fig. 2 Nuclear defects are intrinsic to Lmna KO myonuclei.

a, Top, schematic of the generation of hybrid myofibres containing nuclei from both Lmna WT and Lmna KO cell lines. Bottom, corresponding representative images. Final hybrid fibres contained ~80% Lmna WT nuclei and 20% Lmna KO nuclei. Arrowheads denote Lmna KO nucleus with a chromatin protrusion residing within the same myofibre as a Lmna WT nucleus. Experiments were conducted three independent times, with similar results. b, Quantification of the number of chromatin protrusions from Lmna WT and Lmna KO nuclei contained within isogenic myofibres (control) or hybrid myofibres, typically containing 80% Lmna WT and 20% Lmna KO nuclei. Data points are for n independent experiments, in which 91–163 nuclei were quantified per experiment. Significance determined by two-way ANOVA (nuclear genotype vs. isogenic or hybrid myofibre), using Tukey’s correction for multiple comparisons. All bar plots show mean value ± standard error of the mean.

Extended Data Fig. 3 Nuclear envelope rupture is increased in Lmna N195K myofibres in vitro and in vivo.

Quantification of cGAS-mCherry nuclear envelope rupture reporter foci formation during myoblast differentiation in Lmna N195K a, Lmna H222P b, Lmna cells and wild-type littermate controls (Lmna WT). n = 3 independent experiments per genotype. c, Quantification of the percentage of myonuclei positive for cGAS-tdTomato foci in isolated muscle fibres from Lmna WT, Lmna KO, Lmna N195K, and Lmna H222P mice expressing the cGAS-tdTomato transgene. Analysis performed for whole fibre (left) and by classification of nuclei located at the MTJ or within the body of the fibre (right). Data for Lmna WT and Lmna KO reproduced from Fig. 4e for comparison. Data based on n individual myofibres per genotype, isolated from 4–5 animals each. All bar plots show mean value ± standard error of the mean.

Extended Data Fig. 4 Lmna KO myonuclei with the highest amount of γH2AX foci frequently display chromatin protrusions.

Analysis of DNA damage, assessed by γH2AX staining, in Lmna KO nuclei, comparing nuclei with chromatin protrusions to those without protrusions. Chromatin protrusions were assessed based on the presence of chromatin extending beyond the nuclear envelope, marked by lamin B-staining. Data based on n independent cell lines per genotype. Significance determined by two-tailed students t-test. All bar plots show mean value ± standard error of the mean.

Extended Data Fig. 5 Lmna KO myotubes have no defects in DNA damage repair.

a, Representative images of γH2AX foci in Lmna WT and Lmna KO myotubes at 3, 6 and 24 h following a 5 Gy dose with radiation or no irradiation control. b, Quantification of γH2AX after 3, 6 and 24 h post-irradiation or no irradiation control. Data based on n independent cell lines. Significance determined by two-way ANOVA (genotype; time point), using Tukey’s correction for multiple comparisons. All bar plots show mean value ± standard error of the mean.

Extended Data Fig. 6 Mechanical reinforcement of Lmna KO myonuclei by microtubule stabilization reduces nuclear damage.

a, Representative image of nuclear deformation following microharpoon strain application in Lmna KO myotubes at day five of differentiation. Myotubes were treated for 24 h with either paclitaxel or DMSO control. Yellow dotted line denotes the perimeter of the nucleus prior to strain application. Scale bar: 20 µm. Similar results were obtained in > 10 nuclei in at least three independent experiments (see panel b). b, Quantification of nuclear strain in Lmna WT and Lmna KO myofibres using microharpoon assay following 24 h of treatment with 50 nM paclitaxel or DMSO vehicle control. Data points are from n nuclei per genotype and condition from three independent experiments. Significance determined by two-way ANOVA (genotype; drug treatment), using Tukey’s correction for multiple comparisons. c, Quantification of chromatin protrusions at day 7 of differentiation, following treatment with paclitaxel (50 nM) or DMSO starting at day 4 of differentiation. Data based on n independent experiments per condition. Significance determined by one-way ANOVA, using Tukey’s correction for multiple comparisons. d, Quantification of cGAS-mCherry foci formation during 10 day myofibre differentiation following treatment with paclitaxel (10 nM) or DMSO control, starting at day 5 of differentiation. Data based on n = 3 independent experiments. All bar plots show mean value ± standard error of the mean.

Extended Data Fig. 7 Inhibiting myofibre contractility does not prevent nuclear envelope rupture in Lmna KO myofibres.

a, Quantification of cGAS-mCherry foci formation during 10 day myofibre differentiation follow treatment with nifedipine (5 µM), which inhibits contractility, or DMSO vehicle control, starting at day 5 of differentiation. Data based on three independent experiments. b, Quantification of chromatin protrusions at day 7 of differentiation following treatment with nifedipine (10 µM) or DMSO, starting at day 4 of differentiation. Data points are from n images per genotype and condition, from three independent experiments. Significance determined by one-way ANOVA, using Tukey’s correction for multiple comparisons. All bar plots show mean value ± standard error of the mean.

Extended Data Fig. 8 Kif5b depletion in myotubes reduced chromatin protrusions and DNA damage in Lmna KO myonuclei.

a, Western blot for Kif5b in myoblasts treated with a non-target control siRNA (siRNA NT) or siRNA against Kif5b. Data based on n independent experiments. (Bottom) Corresponding quantification. Significance determined by two-tailed students t-test within each genotype. b, Representative images of Lmna KO myofibres at day 5 of differentiation treated with either a non-target control siRNA (siRNA NT) or siRNA against kinesin-1 (siRNA Kif5b) at day 0. Scale bar: 20 µm. Quantification of the number of chromatin protrusions at day 5 of differentiation in Lmna KO cells treated with non-target (NT) siRNA or depleted for Kif5b using two independent siRNAs (Kif5b #3 and Kif5b #4). Data based on n independent experiments, with 155–270 nuclei counted per experiment. Significance determined by one-way ANOVA, using Tukey’s correction for multiple comparisons. c, Representative images of Lmna KO cells treated with either non-target (NT) siRNA or siRNA against Kif5b and immunofluorescently labeled for γH2AX, showing fewer chromatin protrusions and less DNA damage in the Kif5b depleted cells. Experiments were conducted three independent times, with similar results. Scale bar: 20 µm. d, Quantification of the number of γH2AX foci in Lmna KO myonuclei following treatment with either non-target siRNA or siRNA against Kif5b. Data based on n independent experiments, in which 27–53 nuclei were counted per experiment. All bar plots show mean value ± standard error of the mean. Source data

Extended Data Fig. 9 Expression of the DN-KASH2 construct disrupts the LINC complex and limits nuclear movement, without affecting myofibre function in Lmna WT myofibres.

a, Representative image showing displacement of endogenous nesprin-1 in myofibres expressing the DN-KASH2 construct, and no displacement of nesprin-1 in myofibres expressing the DN-KASH2ext construct. Scale bar: 10 µm. b, Representative image showing nuclear clustering in myofibres expressing the DN-KASH2 construct, and normal nuclear spreading in myofibres expressing the DN-KASH2ext construct. Scale bar: 20 µm. (c) Quantification of cell viability following DN-KASH2 or DN-KASH2ext treatment in Lmna WT cells using the MTT assay. Data based on n differentiation replicates per condition, from three independent experiments. Significance determined by two-way ANOVA (genotype; DOX treatment), using Tukey’s correction for multiple comparisons. d, Quantification of myofibre contraction following DN-KASH2 or DN-KASH2ext treatment in Lmna WT cells based on the percent of contractile fibres. Data based on n independent experiments, for which scores were determined based on 5–6 image sequences per condition. Significance determined by two-way ANOVA (genotype x DOX treatment), using Tukey’s correction for multiple comparisons. e, Representative images of Lmna KO expressing either DN-KASH2 or DN-KASH2ext, with or without 1 µM doxycycline (DOX), and immunofluorescently labeled for myosin heavy chain, actin, and DNA (DAPI) showing increased cell area and enhanced sarcomeric staining in the DOX treated cells expressing DN-KASH2. Scale bar: 50 µm. f, Quantification of the number of chromatin protrusions in Lmna KO myonuclei expressing either DN-KASH2 or DN-KASH2ext. Data based on n independent experiments per condition. Significance determined by two-way ANOVA (genotype; DOX treatment), using Tukey’s correction for multiple comparisons. g, Quantification of the extent of DNA damage based on the number of γH2AX foci per nucleus during myofibre differentiation. Lmna KO myonuclei expressing the DN-KASH2 construct show a decrease in the nuclei with severe DNA damage ( > 25 foci). Data based on n independent experiments per condition. All bar plots show mean value ± standard error of the mean.

Extended Data Fig. 10 Proposed mechanism by which Lmna mutations result in myofibre dysfunction and death.

Kinesin-1 motor proteins spread myonuclei along the myotubes axis during differentiation. In Lmna mutant cells, which have mechanically weaker nuclei, the localized forces associated with nuclear migration cause chromatin protrusion and NE ruptures. This mechanically induced nuclear damage results in DNA damage, detected by γH2AX foci, and activation of DNA damage response pathways, which leads to decline in myofibre health and cell death. b, Schematic flow chart delineating the steps described in panel a, along with interventions explored in this work. Stabilizing microtubules surrounding the myonuclei reinforces the Lmna mutant nuclei and prevents chromatin protrusions and NE ruptures. Inhibiting nuclear movement by Kif5b depletions similarly prevents nuclear damage. Muscle contractions may also contribute to nuclear damage in vivo.

Supplementary information

41563_2019_563_MOESM3_ESM.avi

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation.

41563_2019_563_MOESM4_ESM.avi

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation.

41563_2019_563_MOESM5_ESM.avi

Representative movie of spontaneous contractions in Lmna N195K myofibres after 10 d of differentiation.

41563_2019_563_MOESM6_ESM.avi

Representative movie of spontaneous contractions in Lmna H222P myofibres after 10 d of differentiation.

41563_2019_563_MOESM7_ESM.avi

Representative movie of micropipette aspiration of Lmna WT, Lmna KO, Lmna N195K and Lmna H222P myoblasts.

41563_2019_563_MOESM8_ESM.avi

Representative movie of micro-harpoon manipulation of Lmna WT and Lmna KO myotubes after 5 d of differentiation.

41563_2019_563_MOESM9_ESM.avi

Time-lapse of nuclear envelope rupture in Lmna KO myotubes after 4 d of differentiation. Note the loss of soluble NLS-GFP from the nucleus into the cytoplasm.

41563_2019_563_MOESM10_ESM.avi

Representative movie of micro-harpoon manipulation of Lmna KO myotubes after 5 d of differentiation following 24 h of treatment with either 50 nM paclitaxel or DMSO control.

41563_2019_563_MOESM11_ESM.avi

Time-lapse of NE rupture during myonuclear movement after 5 d of differentiation. Note that the loss of NLS-GFP from the nucleus is immediately followed by the formation of cGAS-mCherry foci at the site of rupture.

41563_2019_563_MOESM12_ESM.avi

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation expressing a doxycycline-inducible GFP-KASH2 to disrupt nucleo-cytoskeletal force transmission. Non-doxycycline-treated control.

41563_2019_563_MOESM13_ESM.avi

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation expressing a doxycycline-inducible GFP-KASH2 to disrupt nucleo-cytoskeletal force transmission. Doxycycline-treated cells expressing GFP-KASH2.

41563_2019_563_MOESM14_ESM.avi

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation expressing the doxycycline-inducible GFP-KASH2ext control. Non-doxycycline-treated control.

41563_2019_563_MOESM15_ESM.avi

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation expressing the doxycycline-inducible GFP-KASH2ext control. Doxycycline-treated cells expressing GFP-KASH2ext.

41563_2019_563_MOESM16_ESM.avi

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation expressing a doxycycline-inducible GFP-KASH2 to disrupt nucleo-cytoskeletal force transmission. Non-doxycycline-treated Lmna KO control.

41563_2019_563_MOESM17_ESM.avi

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation expressing a doxycycline-inducible GFP-KASH2 to disrupt nucleo-cytoskeletal force transmission. Doxycycline-treated Lmna KO cells expressing GFP-KASH2.

41563_2019_563_MOESM18_ESM.avi

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation expressing the doxycycline-inducible GFP-KASH2ext control. Non-doxycycline-treated Lmna KO controls.

41563_2019_563_MOESM19_ESM.avi

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation expressing the doxycycline-inducible GFP-KASH2ext control. Doxycycline-treated Lmna KO cells expressing GFP-KASH2ext.

Supplementary Information

Supplementary Figs. 1–13, Tables 1 and 2 and refs. 1–2.

Reporting Summary

Supplementary Video 1

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation.

Supplementary Video 2

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation.

Supplementary Video 3

Representative movie of spontaneous contractions in Lmna N195K myofibres after 10 d of differentiation.

Supplementary Video 4

Representative movie of spontaneous contractions in Lmna H222P myofibres after 10 d of differentiation.

Supplementary Video 5

Representative movie of micropipette aspiration of Lmna WT, Lmna KO, Lmna N195K and Lmna H222P myoblasts.

Supplementary Video 6

Representative movie of micro-harpoon manipulation of Lmna WT and Lmna KO myotubes after 5 d of differentiation.

Supplementary Video 7

Time-lapse of nuclear envelope rupture in Lmna KO myotubes after 4 d of differentiation. Note the loss of soluble NLS-GFP from the nucleus into the cytoplasm.

Supplementary Video 8

Representative movie of micro-harpoon manipulation of Lmna KO myotubes after 5 d of differentiation following 24 h of treatment with either 50 nM paclitaxel or DMSO control.

Supplementary Video 9

Time-lapse of NE rupture during myonuclear movement after 5 d of differentiation. Note that the loss of NLS-GFP from the nucleus is immediately followed by the formation of cGAS-mCherry foci at the site of rupture.

Supplementary Video 10

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation expressing a doxycycline-inducible GFP-KASH2 to disrupt nucleo-cytoskeletal force transmission. Non-doxycycline-treated control.

Supplementary Video 11

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation expressing a doxycycline-inducible GFP-KASH2 to disrupt nucleo-cytoskeletal force transmission. Doxycycline-treated cells expressing GFP-KASH2.

Supplementary Video 12

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation expressing the doxycycline-inducible GFP-KASH2ext control. Non-doxycycline-treated control.

Supplementary Video 13

Representative movie of spontaneous contractions in Lmna WT myofibres after 10 d of differentiation expressing the doxycycline-inducible GFP-KASH2ext control. Doxycycline-treated cells expressing GFP-KASH2ext.

Supplementary Video 14

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation expressing a doxycycline-inducible GFP-KASH2 to disrupt nucleo-cytoskeletal force transmission. Non-doxycycline-treated Lmna KO control.

Supplementary Video 15

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation expressing a doxycycline-inducible GFP-KASH2 to disrupt nucleo-cytoskeletal force transmission. Doxycycline-treated Lmna KO cells expressing GFP-KASH2.

Supplementary Video 16

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation expressing the doxycycline-inducible GFP-KASH2ext control. Non-doxycycline-treated Lmna KO controls.

Supplementary Video 17

Representative movie of spontaneous contractions in Lmna KO myofibres after 10 d of differentiation expressing the doxycycline-inducible GFP-KASH2ext control. Doxycycline-treated Lmna KO cells expressing GFP-KASH2ext.

Source data

Source Data Fig. 4

Unprocessed Western blots of phopho- and total DNA PK

Source Data Extended Data Fig. 8

Unprocessed Western blots of Kif5b and loading control (tubulin)

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Earle, A.J., Kirby, T.J., Fedorchak, G.R. et al. Mutant lamins cause nuclear envelope rupture and DNA damage in skeletal muscle cells. Nat. Mater. 19, 464–473 (2020). https://doi.org/10.1038/s41563-019-0563-5

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