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

Nature Materials volume 19, pages 464–473 (2020)Cite this article

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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.

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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.

References

  1. de Leeuw, R., Gruenbaum, Y. & Medalia, O. Nuclear lamins: thin filaments with major functions. Trends Cell Biol. 28, 34–45 (2018).

    Article  CAS  Google Scholar 

  2. Davidson, P. M. & Lammerding, J. Broken nuclei—lamins, nuclear mechanics, and disease. Trends Cell Biol. 24, 247–256 (2014).

    Article  CAS  Google Scholar 

  3. Maggi, L., Carboni, N. & Bernasconi, P. Skeletal muscle laminopathies: a review of clinical and molecular features. Cells 5, E33 (2016).

    Article  CAS  Google Scholar 

  4. Emery, A. E. Emery–Dreifuss muscular dystrophy—a 40-year retrospective. Neuromuscul. Disord. 10, 228–232 (2000).

    Article  CAS  Google Scholar 

  5. Zwerger, M. et al. Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling. Hum. Mol. Genet. 22, 2335–2349 (2013).

    Article  CAS  Google Scholar 

  6. Gruenbaum, Y. & Foisner, R. Lamins: nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation. Annu. Rev. Biochem. 84, 131–164 (2015).

    Article  CAS  Google Scholar 

  7. Pimentel, M. R., Falcone, S., Cadot, B. & Gomes, E. R. In vitro differentiation of mature myofibers for live imaging. J. Vis. Exp. 119, e55141 (2017).

    Google Scholar 

  8. Lombardi, M. L. et al. The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton. J. Biol. Chem. 286, 26743–26753 (2011).

    Article  CAS  Google Scholar 

  9. Sullivan, T. et al. Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J. Cell Biol. 147, 913–920 (1999).

    Article  CAS  Google Scholar 

  10. Mounkes, L. C., Kozlov, S. V., Rottman, J. N. & Stewart, C. L. Expression of an LMNA-N195K variant of A-type lamins results in cardiac conduction defects and death in mice. Hum. Mol. Genet. 14, 2167–2180 (2005).

    Article  CAS  Google Scholar 

  11. Arimura, T. et al. Mouse model carrying H222P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies. Hum. Mol. Genet. 14, 155–169 (2005).

    Article  CAS  Google Scholar 

  12. Davidson, P. M. et al. High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells. Lab Chip 19, 3652–3663 (2019).

    Article  CAS  Google Scholar 

  13. Fedorchak, G. & Lammerding, J. Cell microharpooning to study nucleo-cytoskeletal coupling. Methods Mol. Biol. 1411, 241–254 (2016).

    Article  CAS  Google Scholar 

  14. Tan, D. et al. Phenotype–genotype analysis of Chinese patients with early-onset LMNA-related muscular dystrophy. PLoS ONE 10, e0129699 (2015).

    Article  CAS  Google Scholar 

  15. Gnocchi, V. F. et al. Uncoordinated transcription and compromised muscle function in the lmna-null mouse model of Emery–Dreifuss muscular dystrophy. PLoS ONE 6, e16651 (2011).

    Article  CAS  Google Scholar 

  16. Denais, C. M. et al. Nuclear envelope rupture and repair during cancer cell migration. Science 352, 353–358 (2016).

    Article  CAS  Google Scholar 

  17. Raab, M. et al. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death. Science 352, 359–362 (2016).

    Article  CAS  Google Scholar 

  18. Park, S.-J. et al. DNA-PK promotes the mitochondrial, metabolic, and physical decline that occurs during aging. Cell Metab. 25, 1135–1146 (2017). e1137.

    Article  CAS  Google Scholar 

  19. Irianto, J. et al. Nuclear constriction segregates mobile nuclear proteins away from chromatin. Mol. Biol. Cell 27, 4011–4020 (2016).

    Article  CAS  Google Scholar 

  20. Larsen, B. D. et al. Caspase 3/caspase-activated DNase promote cell differentiation by inducing DNA strand breaks. Proc. Natl Acad. Sci. USA 107, 4230–4235 (2010).

    Article  CAS  Google Scholar 

  21. Connolly, P. F. & Fearnhead, H. O. DNA-PK activity is associated with caspase-dependent myogenic differentiation. FEBS J. 283, 3626–3636 (2016).

    Article  CAS  Google Scholar 

  22. Burla, R., La Torre, M., Merigliano, C., Verni, F. & Saggio, I. Genomic instability and DNA replication defects in progeroid syndromes. Nucleus 9, 368–379 (2018).

    Article  CAS  Google Scholar 

  23. Graziano, S., Kreienkamp, R., Coll-Bonfill, N. & Gonzalo, S. Causes and consequences of genomic instability in laminopathies: replication stress and interferon response. Nucleus 9, 258–275 (2018).

    Article  CAS  Google Scholar 

  24. Roman, W. & Gomes, E. R. Nuclear positioning in skeletal muscle. Semin. Cell Dev. Biol. 82, 51–56 (2018).

    Article  CAS  Google Scholar 

  25. Starr, D. A. Muscle development: nucleating microtubules at the nuclear envelope. Curr. Biol. 27, R1071–R1073 (2017).

    Article  CAS  Google Scholar 

  26. Folker, E. S. & Baylies, M. K. Nuclear positioning in muscle development and disease. Front. Physiol. 4, 363 (2013).

    Article  Google Scholar 

  27. Bone, C. R. & Starr, D. A. Nuclear migration events throughout development. J. Cell Sci. 129, 1951–1961 (2016).

    Article  CAS  Google Scholar 

  28. Stewart-Hutchinson, P. J., Hale, C. M., Wirtz, D. & Hodzic, D. Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness. Exp. Cell Res. 314, 1892–1905 (2008).

    Article  CAS  Google Scholar 

  29. Loewer, A., Karanam, K., Mock, C. & Lahav, G. The p53 response in single cells is linearly correlated to the number of DNA breaks without a distinct threshold. BMC Biol. 11, 114 (2013).

    Article  CAS  Google Scholar 

  30. Fidzianska, A. & Hausmanowa-Petrusewicz, I. Architectural abnormalities in muscle nuclei. Ultrastructural differences between X-linked and autosomal dominant forms of EDMD. J. Neurol. Sci. 210, 47–51 (2003).

    Article  Google Scholar 

  31. Park, Y. E. et al. Nuclear changes in skeletal muscle extend to satellite cells in autosomal dominant Emery–Dreifuss muscular dystrophy/limb-girdle muscular dystrophy 1B. Neuromuscul. Disord. 19, 29–36 (2009).

    Article  Google Scholar 

  32. Gupta, P. et al. Genetic and ultrastructural studies in dilated cardiomyopathy patients: a large deletion in the lamin A/C gene is associated with cardiomyocyte nuclear envelope disruption. Basic Res. Cardiol. 105, 365–377 (2010).

    Article  Google Scholar 

  33. Sylvius, N. et al. In vivo and in vitro examination of the functional significances of novel lamin gene mutations in heart failure patients. J. Med. Genet. 42, 639–647 (2005).

    Article  CAS  Google Scholar 

  34. Nikolova, V. et al. Defects in nuclear structure and function promote dilated cardiomyopathy in lamin A/C-deficient mice. J. Clin. Invest. 113, 357–369 (2004).

    Article  CAS  Google Scholar 

  35. Uchino, R., Nonaka, Y. K., Horigome, T., Sugiyama, S. & Furukawa, K. Loss of Drosophila A-type lamin C initially causes tendon abnormality including disintegration of cytoskeleton and nuclear lamina in muscular defects. Dev. Biol. 373, 216–227 (2013).

    Article  CAS  Google Scholar 

  36. Dialynas, G. et al. LMNA variants cause cytoplasmic distribution of nuclear pore proteins in Drosophila and human muscle. Hum. Mol. Genet. 21, 1544–1556 (2012).

    Article  CAS  Google Scholar 

  37. Cho, S. et al. Mechanosensing by the lamina protects against nuclear rupture, DNA damage, and cell-cycle arrest. Dev. Cell 49, 920–935 (2019). e925.

    Article  CAS  Google Scholar 

  38. Chen, S. N. et al. DNA damage response/TP53 pathway is activated and contributes to the pathogenesis of dilated cardiomyopathy associated with LMNA (lamin A/C) mutations. Circ. Res. 124, 856–873 (2019).

    Article  CAS  Google Scholar 

  39. Hatch, E. M. Nuclear envelope rupture: little holes, big openings. Curr. Opin. Cell Biol. 52, 66–72 (2018).

    Article  CAS  Google Scholar 

  40. Irianto, J. et al. DNA damage follows repair factor depletion and portends genome variation in cancer cells after pore migration. Curr. Biol. 27, 210–223 (2017).

    Article  CAS  Google Scholar 

  41. Fortini, P. et al. DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity. Cell Death Differ. 19, 1741–1749 (2012).

    Article  CAS  Google Scholar 

  42. Ferdousi, L. V. et al. More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny. Stem Cell Res. 13, 492–507 (2014).

    Article  CAS  Google Scholar 

  43. Kruiswijk, F., Labuschagne, C. F. & Vousden, K. H. p53 in survival, death and metabolic health: a lifeguard with a licence to kill. Nat. Rev. Mol. Cell Biol. 16, 393–405 (2015).

    Article  CAS  Google Scholar 

  44. Schwartz, L. M. Skeletal muscles do not undergo apoptosis during either atrophy or programmed cell death–revisiting the myonuclear domain hypothesis. Front. Physiol. 9, 1887 (2019).

    Article  Google Scholar 

  45. Cheema, N., Herbst, A., McKenzie, D. & Aiken, J. M. Apoptosis and necrosis mediate skeletal muscle fiber loss in age-induced mitochondrial enzymatic abnormalities. Aging Cell 14, 1085–1093 (2015).

    Article  CAS  Google Scholar 

  46. Lu, D. et al. LMNA E82K mutation activates FAS and mitochondrial pathways of apoptosis in heart tissue specific transgenic mice. PLoS ONE 5, e15167 (2010).

    Article  CAS  Google Scholar 

  47. Wolf, C. M. et al. Lamin A/C haploinsufficiency causes dilated cardiomyopathy and apoptosis-triggered cardiac conduction system disease. J. Mol. Cell. Cardiol. 44, 293–303 (2008).

    Article  CAS  Google Scholar 

  48. Macquart, C. et al. Microtubule cytoskeleton regulates connexin 43 localization and cardiac conduction in cardiomyopathy caused by mutation in A-type lamins gene. Hum. Mol. Genet. https://doi.org/10.1093/hmg/ddy227 (2018).

  49. Gundersen, G. G. & Worman, H. J. Nuclear positioning. Cell 152, 1376–1389 (2013).

    Article  CAS  Google Scholar 

  50. Jung, H. J. et al. Regulation of prelamin A but not lamin C by miR-9, a brain-specific microRNA. Proc. Natl Acad. Sci. USA 109, E423–E431 (2012).

    Article  Google Scholar 

  51. Coffinier, C. et al. Deficiencies in lamin B1 and lamin B2 cause neurodevelopmental defects and distinct nuclear shape abnormalities in neurons. Mol. Biol. Cell 22, 4683–4693 (2011).

    Article  CAS  Google Scholar 

  52. Bulfield, G., Siller, W. G., Wight, P. A. & Moore, K. J. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl Acad. Sci. USA 81, 1189–1192 (1984).

    Article  CAS  Google Scholar 

  53. Shurer, C. R. et al. Physical principles of membrane shape regulation by the glycocalyx. Cell 177, 1757–1770 (2019). e1721.

    Article  CAS  Google Scholar 

  54. Vogler, T. O., Gadek, K. E., Cadwallader, A. B., Elston, T. L. & Olwin, B. B. Isolation, culture, functional assays, and immunofluorescence of myofiber-associated satellite cells. Methods Mol. Biol. 1460, 141–162 (2016).

    Article  Google Scholar 

  55. Dialynas, G. et al. Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway. PLoS Genet. 11, e1005231 (2015).

    Article  CAS  Google Scholar 

Download references

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).

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Author notes

  1. These authors contributed equally: Ashley J. Earle, Tyler J. Kirby, Gregory R. Fedorchak.

Authors and Affiliations

  1. Meinig School of Biomedical Engineering & Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA

    Ashley J. Earle, Tyler J. Kirby, Gregory R. Fedorchak, Philipp Isermann, Jineet Patel, Sushruta Iruvanti & Jan Lammerding

  2. Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Steven A. Moore

  3. Sorbonne Université, Inserm UMRS 974, Center of Research in Myology, Association Institute of Myology, Paris, France

    Gisèle Bonne

  4. Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Lori L. Wallrath

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Contributions

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.

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Correspondence to Jan Lammerding.

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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

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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