Mechanosensing by the Lamina Protects Against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest

Article

Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest

Graphical Abstract Authors Sangkyun Cho, Manasvita Vashisth, Amal Abbas, ..., Roger A. Greenberg, Benjamin L. Prosser, Dennis E. Discher

Correspondence [email protected]

In Brief Progeroid syndromes make affected individuals appear older, and the only known causes are mutations in DNA repair factors and lamin-A. Cho et al. show that as embryonic hearts stiffen and strengthen in development, lamin-A accumulates to oppose stress-driven nuclear rupture, repair factor loss, increased DNA damage, and cell-cycle arrest.

Highlights d Stiff ECM, high actomyosin contractility, and low lamin-A favor nuclear rupture d Nuclear rupture causes cytoplasmic mis-localization of DNA repair factors d Repair factor depletion increases DNA damage, telomere loss, and cell-cycle arrest d Lamin-A increases during development to mechano-protect the genome

Cho et al., 2019, Developmental Cell 49, 920–935 June 17, 2019 ª 2019 Elsevier Inc. https://doi.org/10.1016/j.devcel.2019.04.020 Author Correction: Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell Cycle Arrest Sangkyun Cho1, Manasvita Vashisth1, Amal Abbas1, Stephanie Majkut1, Kenneth Vogel1, Yuntao Xia1, Irena L. Ivanovska1, Jerome Irianto1, Manorama Tewari1, Kuangzheng Zhu1, Elisia D. Tichy2, Foteini Mourkioti2, Hsin-Yao Tang3, Roger A. Greenberg4, Benjamin L. Prosser5, and Dennis E. Discher1,2,4-6*

1 Molecular & Cell Biophysics Lab, University of Pennsylvania, Philadelphia, PA 19104, USA 2 Orthopaedic Surgery and Cell & Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 3 Center for Systems and Computational Biology, Wistar Institute, Philadelphia, PA 19104, USA 4 Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 5 Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 6 Penn Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA * Lead contact: [email protected]

Summary Several figures in this article have been mis-referenced per the accompanying Amendment.

Amendment A number of Figures and Supplementary Figures have been mis-referenced in this article:

• p.6: “…mis-localization persists for hours (Figure 2E-ii)”  Figure S2E-ii • p.9: “…how LMNA adjusts to actomyosin stress (Figures 1A-1E, 2C, and 2E-iii)”  Figure 1A-1E • p.11: “…spreading, and sarcomere assembly (Figures 6C, 5D-ii, S6H)”  Figures 6C, 6D-ii, S6A,E • p.11: “…at the single-cell level (Figure 5D-i)”  Figure 6D-i • p.12: “…distributed throughout the nucleoplasm (Figures 3E-ii and 4D)”  Figures 3E-ii and 4A-i • p.12: “…multiple DNA repair factors in several pathways (Figure 2E-i)”  Figure S2E-i • p.12: “…LMNA of the ‘nuclear matrix’ (Figures 5D-i, 5A, and 5B)”  Figures 5D-i, 6C, and 6D) • p.12: “…intermittently strain the nucleus (Figures 3F, 5D, and S6G)”  Figures 3F, 6D, and S6G • p.13: “…hearts across species (Cho, et al., 2017) (Figure 7A)”  Figure S7D • p.13: “…diverse chick embryo tissues at E18 (Figure 7B)”  Figure S7E • p.13: “…stiffness of a tissue in adults (Figure 7C; adapted from…”  Figure S7F • Acknowledgements: Support of National Science Foundation Materials Research Science and Engineering Center at Penn is gratefully acknowledged.

The findings and conclusions of the article remain unchanged. The original article has not been corrected. Developmental Cell Article

Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest

Sangkyun Cho,1 Manasvita Vashisth,1 Amal Abbas,1 Stephanie Majkut,1 Kenneth Vogel,1 Yuntao Xia,1 Irena L. Ivanovska,1 Jerome Irianto,1 Manorama Tewari,1 Kuangzheng Zhu,1 Elisia D. Tichy,2 Foteini Mourkioti,2 Hsin-Yao Tang,3 Roger A. Greenberg,4 Benjamin L. Prosser,5 and Dennis E. Discher1,2,4,5,6,7,* 1Molecular & Cell Biophysics Laboratory, University of Pennsylvania, Philadelphia, PA 19104, USA 2Orthopaedic Surgery and Cell & Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 3Center for Systems and Computational Biology, Wistar Institute, Philadelphia, PA 19104, USA 4Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 5Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 6Penn Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 7Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.04.020

SUMMARY modulated by the stiffness of the extracellular matrix (ECM), which generally promotes actomyosin stress (Engler et al., Whether cell forces or extracellular matrix (ECM) can 2006; Paszek et al., 2005) and affects the conformation (Sawada impact genome integrity is largely unclear. Here, et al., 2006), post-translational modification (PTM) (Guilluy et al., acute perturbations (1 h) to actomyosin stress or 2014), localization (Dupont et al., 2011), and degradation (Dingal ECM elasticity cause rapid and reversible changes and Discher, 2014) of mechanosensitive proteins. Actomyosin in lamin-A, DNA damage, and cell cycle. The findings links to ECM and to nuclei, such that rapid changes to the are especially relevant to organs such as the heart ECM and tissue mechanics that result from chronic or acute injury or even drug therapies (e.g., cardiac arrest or cardiomyop- because DNA damage permanently arrests cardio- athy treatment [Green et al., 2016]) can in principle affect the nu- myocyte proliferation shortly after birth and thereby cleus (Takaki et al., 2017; Wiggan et al., 2017; Kanellos et al., eliminates regeneration after injury including heart 2015) and perhaps the DNA within. DNA damage and telomere attack. Embryonic hearts, cardiac-differentiated iPS shortening are indeed well documented in injuries that affect cells (induced pluripotent stem cells), and various the heart (Chang et al., 2018; Higo et al., 2017; Sharifi-Sanjani nonmuscle cell types all show that actomyosin- et al., 2017; Oh et al., 2003), as well as nonmuscle tissue—but driven nuclear rupture causes cytoplasmic mis-local- DNA damage and repair are rarely studied in developing organs, ization of DNA repair factors and excess DNA dam- and relationships to proliferation, ECM stiffness, and actomyosin age. Binucleation and micronuclei increase as stress are understudied. telomeres shorten, which all favor cell-cycle arrest. DNA damage and senescence increase with many disease- Deficiencies in lamin-A and repair factors exacerbate linked mutations, including those in the nucleoskeletal protein lamin-A (LMNA) that forms a structural meshwork around chro- these effects, but lamin-A-associated defects are matin (Turgay et al., 2017; Shimi et al., 2015; Gruenbaum et al., rescued by repair factor overexpression and also 2005). LMNA deficiencies associate with elevated DNA damage by contractility modulators in clinical trials. Contrac- (Graziano et al., 2018; Liu et al., 2005) and result in accelerated tile cells on stiff ECM normally exhibit low phosphor- aging of stiff tissues similar to deficiencies in DNA repair factors ylation and slow degradation of lamin-A by matrix- (e.g., KU80) (Li et al., 2007). Moreover, progeroid syndromes are metalloprotease-2 (MMP2), and inhibition of this caused only by mutations in LMNA and multiple DNA repair fac- lamin-A turnover and also actomyosin contractility tors, but LMNA’s primary function in development remains hotly are seen to minimize DNA damage. Lamin-A is thus debated (Burke and Stewart, 2013), with supposed roles in gene stress stabilized to mechano-protect the genome. positioning and regulation (Harr et al., 2015) seeming at odds with largely normal development of human and mouse mutants until weeks after birth. Surprisingly, senescence or apoptosis INTRODUCTION of cells with LMNA defects is rescued by culturing cells on almost any ECM (versus rigid plastic [de La Rosa et al., 2013; Proliferation of many cell types slows dramatically shortly after Hernandez et al., 2010]) and by treatment with at least one birth and is absent in key adult tissues (Li et al., 1996), which pre- drug affecting both cytoskeleton and nucleo-cytoplasmic traf- sents a major challenge to regeneration. Cell growth in culture is ficking (Larrieu et al., 2018, 2014). Relationships between lamins,

920 Developmental Cell 49, 920–935, June 17, 2019 ª 2019 Elsevier Inc. LMNA & DNA damage are mechano-sensitive to myosin-II & matrix, but DNA damage levels change more rapidly

A Embryonic age: Beating E4 heart Day-4 = E4 0.5 h 1 h 0.04 Isolate ref

ref Washout +/- OM 20x hearts Cardiac arrest 0.02 L W + Blebb./MYK AR / AR AR Δ AR / AR / AR DMSO 0.00 Blebb. Aspect Ratio 5 sec AR = L/W Washout lebb. OM B 1h Washout Time (h) Blebb. (25 μM) DMSO Blebb Wash. 0 246 μ Time (h): 3h 2h +1h kDa 1.5 MYK (0.3 M) LMNA 73 C μ i MYK (1 M) γH2AX 17 B LMNB1 66 DMSO Ctrl 1.0 DMSO Blebbistatin Washout DMSO OM +OM 0.6 by Mass Spec. * Time (h): 0.5 0.5 1 2 3 4.5 +1 +3 6 +3 kDa LMNA 73 Washout 0.4 n.s. 0.5 γH2AX 17 ** 0.2 Lamin-A (A.U.) Lamin-A n.s. # # 42 Washout at 1h

Δ 0.0 LMNA γH2AX Myosin-II inhibition Rescue to DMSOnorm. <0.5h,6h> Ctrl 0.0 0 1 2 3 4 5 6 7 8

D ii 1.5

- + * - + DMSO Washout +OM - + 1.0 - + *** DMSO Ctrl - + * Blebb. DMSO MYK (0.3μM)MYK kDa(1μM) - + LMNA 73 n.s. *** - + 42

- + H2AX (A.U.) 0.5 MYK γ γ 17 - + n.s. H2AX 42 - + Ionizing - + Radiation - + Ctrl <0.5h,6h> DMSO to norm. 0.0 0 5 10 15 20 0 1 2 3 4 5 6 7 8 Comet centroid distance (μm) Time (h)

LMNA KD & myosin-II inhibition/washout perturb cell cycle in intact hearts E NT Collagenase (0.3 mg/ml) Collagenase (1 mg/ml) of population TGM F G i AR / AR '2N' '4N' DMSO 2N ref Blebb (2h) 4N S * 0 1 2 3 4 Wash. (+1h) Time (h) 3 sec 10 100 0.06 n.s. ref +Blebb (2h) 100 60 0.04 ns 0.02 G1 G2 40 ns**

TGM AR AR /

Δ *** Col’ase 0.00 20 NT Col’ase TGM MO MO MO of population Ctrl LMNA LMNA EdU intensity (A.U.) Conc.(mg/ml): -- 0.3 1 20 20 20/1 Time (h): 10 0 1 0.75 0.75 0.53 3+0.75 kDa LMNA 0 1 2 3 G1 S G2 73 ii +Blebb (2h) Total DNA (A.U.) MO MO MO γ ctrl LMNA LMNA kDa H2AX 17 LMNA 73 γ 42 H2AX 17 p21 18 Rescue H

42 Matrix stiffness

LMNB1 66 Collagenase (1 mg/ml) HSP90 90 Cytopl. TGM LMNA 2.0 LMNA Acto-myosin Nucleus * γH2AX stress γ 1.5 ~1h 1 * H2AX (A.U.) * n.s. p21 >3h NT Ctl 1 Lamin-A LMNA ctrl n.s. ? * 1.0 * *** * Nuclear DNA norm. to NT Immunoblot Δ Immunoblot 0.5 rupture damage 0.4 0 1 norm. to NTnorm. (A.U.) by Mass Spec. Col'ase (mg/ml) 0 norm. to MO 0.0 0 1 2 3 4 MO MO MO Ctrl LMNA LMNA Cell cycle arrest Time (h) +Blebb (2h)

Figure 1. Contractility or Collagen Perturbations Result in Rapid 1 h Changes in LMNA, DNA Damage, and Cell Cycle (A) Chick hearts from day 4 (E4) beat at 1–2 Hz for up to 5 days. Middle: aspect ratio (AR) beating strain is arrested by myosin-II inhibition but recovers with drug washout ± myosin-II activator, OM. (B) Immunoblot of hearts inhibited for varying durations, followed by washout ± OM (8 hearts per lysate). (legend continued on next page)

Developmental Cell 49, 920–935, June 17, 2019 921 actomyosin stress, ECM mechanics, and DNA damage are reliably. Quantitative immunoblots and mass spectrometry (MS) nonetheless obscure—especially in tissues. measurements (Figures 1B, 1C-i, S1A, and S1B) revealed a rapid Embryonic hearts beat spontaneously for days after isolation decrease in LMNA (45 min decay constant), while LMNB re- from early chick embryos, and beating is acutely sensitive to mained unchanged (Figure 1C-i, upper right inset; Figure 1C), myosin-II inhibition (Figure 1A), as well as enzymatic stiffening and similar results were evident for a cardiac myosin-II-specific or softening of the ECM (Majkut et al., 2013). The latter studies inhibitor mavacamten analog (MYK)—in clinical trials for hyper- reveal an optimal stiffness for beating that is likewise evident trophic cardiomyopathy (National Institutes of Health, 2016a for cardiomyocytes (CMs) cultured on gels (Majkut et al., 2013; [NCT02842242]). Despite rapid recovery of beating after drug Engler et al., 2008; Jacot et al., 2008). DNA damage is conceiv- washout (Figure 1D), LMNA recovered more slowly (>3 h), indi- ably optimized in heart as it triggers a switch from proliferation to cating slow synthesis. senescence in post-natal hearts (Puente et al., 2014). DNA dam- DNA damage in post-natal development with LMNA defi- age is also implicated in telomere attrition and binucleation of ciencies (Liu et al., 2005) led us to hypothesize acute, embryonic CMs that signal irreversible exit from cell cycle (Aix et al., changes in phospho-histone gH2AX, as a primary marker of DNA 2016). We postulated embryonic hearts with rapidly tunable damage. A rapid decrease in DNA damage was surprising with mechanics could prove useful as a tissue model for clarifying myosin-II inhibition (Figure 1C-ii) given the decrease LMNA, but protein-level mechanosensing mechanisms in vivo that could an electrophoretic comet assay confirmed the gH2AX results be studied thoroughly in vitro with many cell types. (Figure 1D). It is useful to keep in mind that the heart beats reason- LMNA is reportedly ‘‘undetectable’’ in early embryos (Stewart ably well with LMNA deficiencies and mutations. Because blebb and Burke, 1987), but it accumulates clearly and progressively in washout recovers beating while LMNA remains low, we antici- tissues such as heart, bone, and lung (Solovei et al., 2013; Ro¨ ber pated a large spike in DNA damage shortly after washout (Figures et al., 1989; Lehner et al., 1987) and is highest in adults within 1C-i and 1C-ii, right inset). LMNA and DNA damage eventually these mechanically stressed, stiff tissues that are collagen rich reached control levels (in hours), but the spike highlights the (Swift et al., 2013). LMNA deficiencies accordingly produce disruptive effects of actomyosin stress on genome integrity. measurable defects weeks after birth in such tissues, including Actomyosin contractility is generally downstream of ECM heart (Kubben et al., 2011; Worman and Bonne, 2007). We there- stiffness (Ulrich et al., 2009; Engler et al., 2006), including for fore hypothesized that LMNA in normal embryos mechano- immature CMs (Engler et al., 2008; Jacot et al., 2008). Acute per- senses the earliest microenvironment in the first organ and turbations of collagen matrix might therefore be expected to increases not only to stiffen the nucleus (Osmanagic-Myers affect DNA damage. Collagenase treatment for 45-min indeed et al., 2015; Dahl et al., 2008; Pajerowski et al., 2007) but also resulted in rapid decreases in DNA damage and LMNA (Fig- to regulate repair factors that confer resistance to both DNA ure 1E), consistent with rapid softening of E4 hearts (50%) damage and cell-cycle arrest. and weaker beating (Majkut et al., 2013). Treatment with tissue transglutaminase (TGM), a cross-linker of ECM that stiffens RESULTS heart and thereby increases basal tension (>2-fold in 2 h [Majkut et al., 2013]), increased gH2AX and LMNA (only after 3 h) except Contractility and Collagen Perturbations Rapidly Impact when collagenase was subsequently added (Figure 1E). LMNA LMNA and DNA Damage in Hearts thus decreases quickly or increases slowly in response to To first assess the consequences of cytoskeletal stress on nuclei changes in ECM stiffness or actomyosin tension, both of which in a tissue, we arrested embryonic hearts (embryonic day [E] 4) appear to also affect DNA damage. Effects are also generally with reversible inhibitors of myosin-II (Figure 1A). Spontaneous reversible. beating of hearts stopped in minutes with blebbistatin (blebb), but the heart re-started just as quickly upon washout of DNA Damage in LMNA-Deficient Hearts Perturbs Cell drug (Figure 1A, right). An activator of cardiac myosin-II omecam- Cycle and Causes Aberrant Beating tiv mecarbil (‘‘OM’’)—in clinical trials for heart failure (National In- Excess DNA damage has been shown to impact the cell cycle stitutes of Health, 2016b [NCT02929329])—re-started the hearts in post-natal CMs (Puente et al., 2014), and so, we next sought

(C) (i) Immunoblot densitometry reveals LMNA decreases in 45-min upon myosin-II inhibition, but LMNA recovery takes >3 h upon washout. MS confirms 1-h data with 29 LMNA-specific peptides, and inset shows LMNB1 remains unchanged. (ii) DNA damage gH2AX also decreases but spikes upon blebb washout (5.5 h, light blue). Bar graph of LMNA and gH2AX changes 1 h post washout. Bottom right immunoblot shows MYK treatment for 1.5 h also affects LMNA and gH2AX. One-way ANOVA (Dunnett’s test versus DMSO Ctrl avg of 0.5 h, 6 h): *p < 0.05, ** < 0.01; t test (versus plateau average of 2, 3, and 4.5 h time points): #p < 0.05. All error bars indicate ± SEM. (D) Electrophoretic comet assay of DNA damage validates gH2AX quantitation, with g-irradiation control. (E) Immunoblot of hearts treated with collagenase and/or transglutaminase (TGM) (8 hearts per lysate). Matrix digestion rapidly decreases LMNA and gH2AX, consistent with low stress; cross-linking has opposite and reversible effect by 3 h (t test: *p < 0.05; superfluous blot lanes have been removed and indicated by a blank space).

(F) (i) Morpholino KD of LMNA (MOLMNA) does not affect beating versus MOCtrl, but (ii) MOLMNA increases DNA damage (gH2AX) and cell cycle inhibitor p21 unless rescued by myosin-II inhibition (i and ii). Two immunoblots (anti-LMNA, p21, and b-actin) and display proteins (anti-LMNB1, gH2AX, HSP90) of interest above loading controls. (8 hearts per lysate). One-way ANOVA: *p < 0.05, *** < 0.001. (G) (i) Representative plot of EdU versus DNA intensity for cell cycle phase (G1 versus S versus G2; or 2 N versus 4 N). (ii) Blebb washout increases G2 and 4 N cells (versus 2 N). n > 77 cells per condition; t test: **p < 0.01. (H) Proposed mechanosensing pathway.

922 Developmental Cell 49, 920–935, June 17, 2019 AB

Figure 2. DNA Damage in Embryonic Hearts Causes Aberrant Beating (A) 1 h etoposide reversibly increases DNA damage 4-fold (8 hearts per group). All error bars indicate ± SEM.

(B) Beating (top) after 1 h of treatment is only affected by etoposide. (i and ii) % hearts exhibiting arrhythmia. Oxidant H2O2 shows distinct kinetics. (iii and iv) Beating decreases and arrests compared to DMSO. 8–16 hearts per grp; t test *p < 0.05, ** < 0.01, *** < 0.001. (C) Schematic: the level of DNA damage that results from various sources depends also on levels of repair factors in the nucleus.

to assess the biological consequences of DNA damage in assessed DNA damage that was directly and abruptly increased LMNA-suppressed embryonic hearts. Morpholino (MO) medi- with etoposide (4-fold, 1 h) (Figure 2A). This inhibitor of replication ated knockdown (KD) of LMNA (MOLMNA; 40% KD in 24 h) and transcription quickly caused aberrant beating that could be was achieved with no signiﬁcant effect on contractile beating reversed upon drug washout (Figure 2B); many cardiac laminopa- (Figures 1F-i and S1E). LMNA is thus not primarily upstream of thies exhibit arrhythmias and broader conduction defects (Fatkin beating, consistent with knockout mice (Singh et al., 2013). et al., 1999). No such effects were seen with the same dose of

Although past studies also suggest LMNA is not detectable in H2O2 (for oxidative stress) relative to DMSO control hearts unless early embryonic hearts and is therefore dispensable (Solovei H2O2 exposure was sustained for days. DNA damage can have et al., 2013; Stewart and Burke, 1987), morpholino KD increased multiple causes, ranging from drugs and oxidative stress to nucle- gH2AX in E4 hearts (Figure 1F-ii). Similar effects were seen by ases, but reversal of damage from natural sources or etoposide in modest repression with retinoic acid (RA) of LMNA (20% only af- hours (Figures 1F, 1G, and 2A) suggest an important role for DNA ter 3 days) that acts transcriptionally (Swift et al., 2013), while RA- repair factors (Figure 2C). antagonist (AGN) upregulated LMNA and suppressed DNA damage (Figures 1F and 1G). blebb was sufﬁcient to rescue the DNA LMNA KD in Intact Hearts and Human iPS-CMs: Nuclear damage in MOLMNA KD hearts, supporting our hypothesis that Rupture and Loss of DNA Repair high contractility coupled to low LMNA causes excess damage. To address how increased mechanical stress causes DNA dam- Bona ﬁde DNA damage can perturb cell cycle (Puente et al., age, the integrity of the nucleus was scrutinized in intact E5 2014); the cell cycle inhibitor p21 indeed trended with gH2AX in hearts doubly transfected with DNA repair protein GFP-KU80 MO-treated hearts (Figure 1F-ii). EdU (5-ethynyl-20-deoxyuridine) (XRCC5) and with a cytoplasmic protein that can bind nuclear integration before and after blebbistatin addition and washout DNA, mCherry-cGAS (cyclic GMP-AMP synthase [Raab et al., (per Figures 1A–1C) further revealed an increase in cells in G2, 2016]) (Figure 3A-i). Cytoplasmic mis-localization of KU80 and with fewer cells in S, and an increase in 4 N cells (Figure 1G). Given concomitant formation of cGAS puncta at the nuclear periphery that acute DNA damage can inhibit cell cycle in developing heart provided evidence of nuclear rupture in transfected hearts (Fig- and is somehow coupled to matrix-myosin-lamins (Figure 1H), we ure 3A-i, yellow arrow), and the fraction of such double-positive

Developmental Cell 49, 920–935, June 17, 2019 923 A D

Figure 3. LMNA Knockdown in Intact Hearts and in hiPS-CMs: Nuclear Envelope Rupture and Loss of DNA Repair Factors (A) (i) Images of E4 hearts transfected with GFP-KU80 and mCherry-cGAS. Arrow: cell with low LMNA and ruptured nucleus, with cytoplasmic mis-localization of GFP-KU80 and mCherry-cGAS puncta at nuclear envelope. (ii) LMNA KD increases mis-localized KU80 and cGAS but is rescued by myosin-II inhibition. (iii) Images of striation (a-actinin-2) before blebb 2 h after and upon washout (1 h). (iv) %-ruptured nuclei (double +’s) decreases with myosin-II inhibition but increases upon washout. Mis-localized KU80 increases further with Imp-i ivermectin (n > 111 double-transfected cells per condition; t test: *p < 0.05). All error bars indicate ±SEM. (B) (i and ii) anti-gH2AX and (i and iii) anti-LMNA immunoblot of blebb-treated E4 hearts after washout ± Imp-i (8 hearts per lysate; t test: *p < 0.05; superﬂuous blot lanes have been removed and is indicated by a blank space).

(C) LMNA immunoﬂuorescence with MOLMNA ± blebb. (n > 139 cells per cond.). (D) Human iPS-derived cardiomyocytes (hiPS-CMs) on stiff 40-kPa gels for 24 h, showing cytoplasmic KU80 in cells with nuclei having LMNA-rich, LMNB- depleted blebs at high curvature sites (arrows). (E) (i and ii) hiPS-CM on soft 0.3-kPa gels minimizes rupture. On rigid plastic, siLMNA KD (40%, n > 119 cells per cond. ([iii]) increases mis-localized KU80 (i) and DNA damage by gH2AX foci (right images). (n > 80 cells per cond.; Dunnett’s test versus NT, i.e. nontreated Ctrl) (F) (i) Passive cyclic stretch of LMNA-knockdown cells expressing GFP-KU80 (8% strain, 1 Hz) increases (ii) %-rupture with cytoplasmic KU80 (upper inset) and (iii) gH2AX. (n > 55 cells per cond.; t test: #p < 0.05, ## < 0.01). (Unless noted otherwise, one-way ANOVA: *p < 0.05, ** < 0.01, *** < 0.001)

cells increased with MOLMNA KD unless blebb was added (Fig- apparent changes in cell viability (Figure 2A-i). DNA damage ure 3A-ii). Myosin-II inhibition disrupted sarcomeres, which again spiked upon blebb washout and remained excessively rapidly recovered upon drug washout (Figure 3A-iii), consistent high with addition of a nuclear import inhibitor (Imp-i; ivermectin) with beating (Figures 1A–1C); and mis-localization of KU80 and that keeps KU80 mis-localized to cytoplasm (Figures 3B-i, 3B-ii, cGAS showed the same trends (Figure 3A-iv), without any and S2A-ii). LMNA immunoﬂuorescence (IF) for MOLMNA and

924 Developmental Cell 49, 920–935, June 17, 2019 blebb/washout hearts (Figures 3B-iii and 3C) confirmed immu- Loss of Repair Factors or LMNA: DNA Damage, noblot trends (Figures 1C-i and 1F) and showed Imp-i kept Binucleation, Micronuclei, and Cell-Cycle Arrest LMNA levels low upon washout. To assess the impact of a limited capacity for DNA repair Human induced-pluripotent-stem-cell-derived CMs (hiPS- in cells, key individual DNA repair factors were partially CMs) cultured on soft or stiff gels with collagen-ligand depleted individually and in combination (si-Combo). BRCA1 constitute another model for clarifying mechanisms at the is implicated in myocardial infarction and ischemia (Shukla single-cell level. Nuclei in hiPS-CMs ‘‘beat’’ (Figure 2B) and et al., 2011), and RPA1 appeared abundant and constant express low LMNA relative to some well-studied human cell in level in our MS of drug- or enzyme-perturbed hearts (Figures lines (e.g., lung-derived A549 cells) and relative to mature 1C and 1E). DNA damage increased in hiPS-CMs for all heart, which is much stiffer than embryos (Swift et al., 2013). repair factor KDs, and the combination proved additive (Fig- Cell morphologies and sarcomere striations resemble those ure 4A-i). of embryonic or fetal CMs. On very stiff gels (40 kPa) but not Excess DNA damage in post-natal hearts has been associated on gels as soft as embryos (0.3 kPa), hiPS-CMs exhibited with CM binucleation (Aix et al., 2016)—a hallmark of irreversible cytoplasmic, mis-localized KU80 (Figure 3D), consistent with cell cycle exit. Soft ECM suppressed binucleation of embryonic envelope rupture under high nuclear stress (Figure 1H). Nuclear CMs relative to stiff ECM (Figure 4A-ii) while suppressing mis- blebs typical of ruptured nuclei formed at points of high localization of repair factors (Figure 3E-i). Partial KD of DNA curvature in such cells (Xia et al., 2018), appearing rich in repair factors directly increased the fraction of binucleated LMNA but depleted of LMNBs (Figure 3D). Stress-induced hiPS-CMs, as well as those with micronuclei (Figure 4A-iii), nuclear rupture was also imaged as rapid and stable accumu- both of which correlated strongly with gH2AX. Repair factor lation of mCherry-cGAS upon nuclear probing with a high cur- KD further increased the fraction of ‘‘4N’’ cells (versus ‘‘2 N’’; Fig- vature atomic force microscopy (AFM) tip (<1 mm), using nano- ures 4A-iv and S3A), in agreement with trends for blebb-treated Newton forces similar to those exerted by a cell’s actomyosin hearts (Figure 1G) and with mitotic arrest. LMNA KD had all of the (Figure 2C). same effects, consistent with a role in repair factor retention in KD of LMNA (siLMNA) doubled the fraction of cells with cyto- nuclei, and, consistent also with a late checkpoint, KD showed plasmic KU80 (Figures 3E-i and S2D-i) and increased gH2AX foci more DNA damage per DNA in 4 N cells relative to control cells (Figures 3E-ii and S2D-ii). These observations seem consistent (Figure 3B). Importantly, with siLMNA or siCombo KD, increased with iPS-CM results for LMNA defects, which show nuclear bleb- gH2AX and cell cycle perturbations were evident not only at bing (Lee et al., 2017), focal loss of nuclear membrane (Siu et al., steady state but also within 1 h of damaging DNA with etoposide 2012), cytoskeletal defects (Bollen et al., 2017), and fibrosis, all of (per Figure 2)—which partially recovered after drug washout which increase with electrical stimulation of contraction. We (Figure 4B). found LMNA levels in hiPS-CMs exhibited sensitivity to substrate Myosin-II inhibition once again rescued the DNA damage, as stiffness similar to that in embryonic hearts (Figure 3E-iii), and we well as cell cycle effects, in siLMNA KD cells (Figure 4C-i), saw blebb-treated siLMNA cells on rigid plastic rescued nuclear whereas siCombo cells maintained high DNA damage (Fig- rupture (Figure 3E-i), as well as DNA damage (Figure 3E-ii). ure 3C). Furthermore, overexpression of relevant DNA repair Relaxation of rigidity-driven actomyosin stress can thus limit nu- factors (with GFP-KU70, KU80, BRCA1 = ‘‘GFP-Combo’’) clear rupture. rescued only the excess DNA damage in nuclear-ruptured cells Cytoplasmic mis-localization of KU80 is merely representa- compared to non-ruptured (Figure 4C-ii). A baseline level of tive: repair factors 53BP1 and RPA2 also simultaneously mis- damage in the latter cells was not affected, likely because repair localized to the cytoplasm upon LMNA KD (Figure 2E-i). Time- factors become limiting only upon nuclear rupture or depletion. A lapse images of GFP-53BP1-expressing cells further revealed combination of factors (GFP-Combo) rescued the excess dam- mis-localization persists for hours (Figure S2E-ii). However, age, while one repair factor had no effect on its own (53BP1) total KU80 levels did not vary (Figure 2F), suggesting that even on the ruptured nuclei. Rescue of DNA damage again sup- nuclear retention is key. pressed the fraction of 4 N cells (Figure 4B lower) and cells in G2 To assess whether mis-localization of repair factors is a gen- (Figure 3D). eral consequence of increased mechanical stress, LMNA KD Since cardiac telomere attrition has been implicated in CM bi- of both CM and U2OS osteosarcoma cells was followed by nucleation (Aix et al., 2016), as well as in cardiomyopathies and cyclic stretch at a frequency and magnitude similar to that of heart failure (Sharifi-Sanjani et al., 2017), we assessed rupture- the intact heart (8% uniaxial strain at 1-Hz frequency) (Fig- associated telomere shortening as a more localized form of DNA ure 3F-i). Mis-localized GFP-KU80 increased >2-fold with damage (Figure 4D). U2OS osteosarcoma cells with ruptured imposed stretching of MOLMNA KD E4 CMs but not MOCtrl (Fig- nuclei (cytoplasmic anti-KU80) showed lower total intensity of ure 3F-ii), and the fold-increase was even more dramatic with telomeric repeat-binding factor-1 (TRF1, as an mCherry fusion; the nonbeating U2OS cells because baseline mis-localization Figure 4E-i) and smaller TRF1 foci size (Figure 4E-ii), as well as was very low. DNA damage also increased with stretching of higher gH2AX in these LMNA KD cells. Importantly, partial KD of MOLMNA CMs while LMNA levels showed an insignificant in- either KU80 or multiple repair factors also caused telomere crease (Figure 3F-iii). An acute increase in stress without a pro- attrition in cells with normal LMNA levels (Figure 4F). Proper portionate increase in LMNA can thus cause mis-localization of retention of DNA repair factors within stressed nuclei thus re- repair factors and DNA damage, which could affect tissue-level quires sufficient LMNA, but LMNA also interacts with key repair function (Figures 2A and 2B). Etoposide-treated hiPS-CM orga- factors (including KU70/80 and RPA1) as confirmed by immuno- noids indeed show aberrant beating (Figure 2G). precipitation-MS (IP-MS) (Figure S3E). IP-MS further indicates

Developmental Cell 49, 920–935, June 17, 2019 925 A B

D E F

GH I

Figure 4. Knockdown of LMNA or DNA Repair Factors Increases DNA Damage and Perturbs Cell Cycle (A) KD of LMNA or repair factors (KU80, BRCA1, RPA1, or si-Combo) in hiPS-CMs increases: (i) gH2AX (ii,iii) binucleated CMs (suppressed on soft matrix, inset) and micronuclei, and (iv) 4 N cells (n > 77 cells per cond.; all error bars indicate ± SEM) (B) KDs increase gH2AX before, during, and after 10 mM etoposide (n > 39 cells per cond.). (C) (i) Blebb rescues siLMNA-induced DNA damage and 4 N cells (n > 400 cells per cond.). (ii) Overexpression of DNA repair factors (GFP-KU70 + KU80 + BRCA1 = GFP-Combo) rescues excess DNA damage (top) and 4 N (bottom) in siLMNA cells only in ruptured nuclei (n > 296 cells per cond.; t test: **p < 0.01, *** < 0.001). (D) Schematic: effect of repair factor mis-localization on telomeres. (E) U2OS expressing mCherry-TRF1. Ruptured nuclei (n = 7) with cytoplasmic KU80 exhibit (i) lower TRF1 and (ii) smaller TRF1 foci (t test: *p < 0.05). (F) siKU80 and si-Combo decrease TRF1 (n > 89 cells per cond.). (G) CoIP-MS of WT LMNA, phosphorylated LMNA (anti-S22), and phospho-mimetic LMNA (GFP-S22E). (H) Cartoon: LMNA-KU80-telomere interactions at the lamina.

(I) Telomere Q-FISH in embryonic CMs shows MOLMNA shortens telomere unless rescued by blebb (n > 81 nuclei per cond.). (Unless noted, one-way ANOVA: *p < 0.05, *** < 0.001)

926 Developmental Cell 49, 920–935, June 17, 2019 ABC

D E

H G I

(legend on next page)

Developmental Cell 49, 920–935, June 17, 2019 927 KU80 interacts preferentially with non-phosphorylated LMNA Collagen-I (a-1 and a2) exhibited the largest fold change in (GFP-wild type [WT]) compared to its phospho-solubilized MS analyses of heart development (Figure 5B), with calibrated nucleoplasmic form (per IP by anti-pSer22 or phosphomimetic spike-ins of purified collagen-I matching past measurements of mutant GFP-S22E) (Figure 4G), suggesting that LMNA scaf- collagen in developing chick hearts (Woessner et al., 1967) folds the repair of DNA at the periphery—which often includes (Figure 4E). Collagen-I becomes the most abundant protein in telomeres (Figure 4H). Importantly, analysis of telomeres in developed animals and is a major determinant of tissue stiff- embryonic CMs by quantitative FISH (Q-FISH) revealed LMNA ness (Shoulders and Raines, 2009); therefore, its increase KD causes telomere shortening unless actomyosin is inhibited together with many other structural proteins (Figure 4C) is (Figure 4I). The findings motivate deeper mechanistic insights consistent with heart stiffening (Figure 2F) (Majkut et al., into how LMNA adjusts to actomyosin stress (Figures 1A–1E, 2013). Mechanosensitive proteins at the interface between ad- 3C, and 3E-iii) to thereby regulate DNA damage and the hesions and actomyosin cytoskeleton were also upregulated, cell cycle. including paxillin (Zaidel-Bar et al., 2007) and vinculin (Huang et al., 2017), consistent with increased adhesion and contractile LMNA Effectively Stiffens Nuclei during Development stress (Geiger et al., 2009). Downregulation of protein synthesis and Increases with Collagen-I ECM and catenins suggest a transition from a rapidly growing, To begin to assess in normal beating hearts how LMNA epithelial-like embryo to a more specialized, terminally differen- senses stress, nuclear deformations were imaged at E4— tiated state. just 1 to 2 days after the first heartbeat (Figure 5A). Transfec- Lamin-A:B ratio increased with collagen-I (Figures 5C-i and tion of a minor fraction of CMs in hearts with mCherry-histone- S4F) and with tissue stiffness Et (Figure 5-ii), with a power law H2B or GFP-LMNA facilitated imaging, and each 1-Hz exponent a = 0.3 (versus collagen-I) similar to that for diverse tis- contraction of heart tissue was seen to strain individual CM sue proteomes (Cho et al., 2017). Experiments fit a ‘‘use it or lose nuclei by up to 5%–8%. Recall that 8% stretch at 1-Hz it’’ mathematical model (Figure 5C-i) wherein mechanical tension was sufficient to rupture CM nuclei (Figure 3F). While transfec- on filamentous collagen-I and LMNA (Dingal and Discher, 2014) tion did not affect overall tissue-level beating, GFP-LMNA suppresses protease-driven degradation (Figure 5C-i). The plot nuclei deformed much less than neighboring control nuclei versus Et yielded a0.7 that is similar for adult tissues (a0.6 (Figure 5A-ii), consistent with in vitro studies showing LMNA [Swift et al., 2013]), with soft E10 brain and stiff E10 liver fitting stiffens the nucleus (Pajerowski et al., 2007; Lammerding well (Figures 4F and 4G). Importantly, 45 min collagenase soft- et al., 2006). MS of lysates quantified 29 unique LMNA pep- ened E4 hearts by 50% and decreased LMNA (Figure 5C), tides at E4 (Figure 1C), confirming our early detection of func- per Figures 1A–1E. tional LMNA (Figure 1B) despite past reports (Solovei et al., Different combinations of drugs were used to identify mecha- 2013; Stewart and Burke, 1987), showed a rapid increase in nisms for how LMNA levels change in tight coordination with LMNA expression by 3-fold from soft E4 hearts to stiffer collagen-I and tissue stiffness. MS-derived heatmaps of drug- E10 hearts (Figures 5B and 5C). B-type lamins remained rela- treated E4 hearts (e.g., collagenase and blebb) reveal rapid tively constant, in agreement with studies of diverse soft and and large decreases in collagen-I, vinculin, and LMNA (Figure 5D; stiff adult tissues that suggest LMNA-specific mechanosensi- confirmed by immunoblot, Figure 5A). Little to no change was tivity (Figures 4A–4C) (Swift et al., 2013). Trends are confirmed observed across most of the detected proteome including by confocal IF and immunoblots of embryonic heart (Figures DNA repair factors (KU70 and RPA1 [Figure 5B-i]), matrix-metal- 5C-i and 5C-ii, insets; Figure S4D). loproteinase MMP2 (pro- and cleaved forms; Figures 5B-ii and

Figure 5. Early Embryonic LMNA Increases with Collagen-I and Tissue Stiffness Et (A) (i) E4 heart beating with GFP-LMNA transfection shows nuclear ‘‘beating.’’ Right: trace of tissue strain tensor quantified using fiduciary transfected nuclei per (Taber et al., 1994) or nuclear strain from nuclear area and aspect ratio (AR). (ii) Overexpression does not affect local or global tissue strain (left), but GFP-LMNA nuclei deform less than mCherry-H2B nuclei (n > 8 nuclei per cond.; t test: *p < 0.05). All error bars indicate ± SEM. (B) Proteomic heatmap of E4, E6, and E10 hearts (n = 8, 2, and 1 heart(s) per lysate, respectively), ranked by fold change from time-average: collagen-I and LMNA top the list. (C) (i) Lamin-A:B ratio from MS scales with collagen-I and fits ‘‘use it or lose it’’ model of tension-inhibited turnover of structural proteins, with stress sensitivity denoted as x. Inset: confocal images of LMNA and B in hearts taken at same settings suggest LMNA increases while LMNB remains constant. (ii) Lamin-A:B scaling versus Et from micropipette aspiration (lower inset), with exponent a similar for adult tissue (a 0.6) (Swift et al., 2013). Cyan: softening by collagenase decreases LMNA. Upper immunoblot (norm. to HSP90): LMNB varies by < 20% from E4-E10 as LMNA increases (n > 8 hearts per lysate). (D) MS intensity fold changes relative to DMSO for Trx-i inhibitor of translation (cycloheximide, 1 mM); MMP-i pan-MMP inhibitor (GM6001, 10 mM); CDK-i (RO3306, at >3.5 mM doses to inhibit many CDKs) to suppress LMNA phosphorylation. Cyan boxes indicate rescues (n = 8 hearts per lysate; t test versus DMSO: *p < 0.05, ** < 0.01, *** < 0.001). (E) (i) E4 hearts treated with blebb ± MMP-i’s (2 h; n = 6 hearts per lysate). pSer390 bands at intact MW (73 kDa) and 42 kDa, consistent with fragments (Baghirova et al., 2016; Prudova et al., 2010) predicted in silico (Song et al., 2012). (ii) Decreased LMNA with blebb is rescued by MMP-i’s, including MMP2-specific ARP-100. (iii) Blebb increases pSer390 of 73 kDa band. (iv) Blebb does not affect pSer390 ratio of 42 kDa and 73 kDa bands, except with MMP-i. (n = 7 hearts per lysate; one-way ANOVA: *p < 0.05, **p < 0.01). (v) Turnover schematic. (F) MS validation of LMNA degradation. (G) Neither blebb nor MMP-i affects catalytic activation of MMP2 (n = 8 hearts per lysate). (H) IF shows nucleoplasmic MMP2 in embryonic CMs is unaffected by blebb. (I) MMP-i rescues blebb-induced decrease in LMNA but does not further suppress gH2AX (n = 6 hearts per lysate). gH2AX is further decreased with CDK-i to inhibit LMNA phosphorylation (n = 8 hearts per lysate; one-way ANOVA: *p < 0.05, ** < 0.01; t test: #p < 0.05).

928 Developmental Cell 49, 920–935, June 17, 2019 A B

D E

Figure 6. LMNA in Isolated Embryonic CMs Is Sensitive to Substrate Stiffness and Actomyosin Stress (A) Airyscan images of embryonic heart. (B) CM cultures on PA gels of controlled stiffness (0.3–40 kPa) and constant collagen-I ligand. (C) Well-separated E4 CMs at 24 h on gels. As with tissue (A), isolated CMs spread, elongate, and striate more with stiffness. Blebb on rigid plastic causes rounded or dendritic CM, with loss of striation. MMP inhibitors have no effect. (legend continued on next page) Developmental Cell 49, 920–935, June 17, 2019 929 5B-iii), cardiac myosin-II (MYH7B), vimentin (VIM), and B-type neonatal CMs (Baghirova et al., 2016; Kwan et al., 2004) sug- lamins. Similar results were obtained even in the presence gests that, independent of maturation stage, active MMP2 is of the translation inhibitor (Trx-i) cycloheximide, except for de- localized appropriately for degradation of phospho-LMNA creases in a few cytoskeletal proteins that indicate rapid transla- downstream of actomyosin tension (Figure 5E-v). tion (e.g., actin [Katz et al., 2012]). Decreased translation thus DNA damage was assessed after acute 2-h treatments of does not explain the heart’s decreases in collagen-I and LMNA CDK-i or MMP-i plus blebb. Whereas MMP-i had no effect on with blebb and/or collagenase. gH2AX relative to blebb alone, CDK-i plus blebb greatly sup- To clarify how myosin-II inhibition (intracellular) leads to a rapid pressed DNA damage (Figure 5I). Since CDK-i maintains decrease in collagen-I (ECM) in intact hearts, a pan-inhibitor of LMNA at the lamina (unlike MMP-i) and thereby stiffens and sta- matrix-metalloproteinases (MMP-i) was used with blebb. The bilizes the nucleus (per Figure 5A and single-cell measurements combination surprisingly prevented collagen-I degradation. In [Buxboim et al., 2014]), the results suggest that even low levels of contrast, a CDK inhibitor (CDK-i; likely affecting CDK4/6) that nuclear stress are resisted to minimize both rupture and DNA limits interphase phosphorylation of LMNA (e.g., pSer22 nor- damage in the embryonic heart. malized to total LMNA: pSer22/LMNA in Figure 5A) rescued blebb-induced decreases in LMNA levels without affecting Isolated CMs Elucidate Effects of ECM Elasticity and the collagen-I decrease. Vinculin’s decrease concurs with Contractility on LMNA Turnover by MMP2 decreased actomyosin tension regardless of MMP-i (Figure 5D), Intact heart presents many complications to understanding but MMP-i surprisingly rescued blebb-induced decreases in LMNA regulation and its function. As the heart stiffens in devel- LMNA similarly to CDK-i (Figures 5D and S5A). opment, for example, collagen ligand also increases greatly for adhesion (Figures 5B and 5C), sarcomere assembly becomes Mechano-protection of the Genome Is Limited by LMNA very dense (Figures 6A and S6A-i), cells and nuclei elongate (Fig- Phospho-solubilization ures 6A-ii and S6B) with stabilization by microtubules (Robison We hypothesized that one or more MMPs directly degrade et al., 2016), cells align (Figure 6C), and nuclear volumes LMNA. Nuclear MMP2 is well documented for many cell types decrease (Figure 6D). Gels with constant collagen-I ligand and including CMs (Xie et al., 2017), and LMNA (but not B-type lam- of controlled stiffness (0.3–40 kPa) (Figure 6B) showed—after ins) has been speculated to be a proteolytic target (Baghirova just 24 h—the same stiffness-correlated morphology and cyto- et al., 2016). Beating E4 hearts treated with MMP-i or an skeleton trends for isolated E4 CMs as the intact heart from E4 MMP2-specific inhibitor, MMP2-i, rescued the blebb-induced to E11 (Figure 6C). Although matrix elasticity had large effects decrease in LMNA in immunoblots that were also probed with on size, shape, sarcomere assembly, and contractility of early a previously untested anti-pSer390 (Figures 5E-i and 5E-ii). For CMs (Figures 6A-i, 6A-ii, and S6E) similar to later stage CMs other phosphosites, interphase phosphorylation, solubilization (Ribeiro et al., 2015; Engler et al., 2008; Jacot et al., 2008), blebb (Kochin et al., 2014), and subsequent degradation of LMNA quickly disrupted cell spreading, striation, and elongation on stiff (Naeem et al., 2015; Bertacchini et al., 2013) are already known gels (10 kPa) and on collagen-coated rigid plastic (Figure 6C). to be suppressed by actomyosin tension on nuclei (Buxboim Gels that mimic the stiffness of E4 heart (1–2 kPa) were optimal et al., 2014; Swift et al., 2013). Intact phospho-LMNA (73 kDa) for cell beating (Figure 6F), consistent with past studies (Majkut increased upon blebb treatment (Figure 5E-iii; pSer390intact/ et al., 2013; Engler et al., 2008), and nuclear ‘‘beating strain’’ in LMNAintact), but even more dramatic was the suppression by isolated CMs (Figures 6F-i and S6G-i) was similar in magnitude MMP2-i and MMP-i (regardless of blebb) of a 42 kDa pSer390 to that in intact heart (5%–8%) (Figure 5A). Surprisingly, lamin- fragment (pSer390fragm./pSer390intact)(Figure 5E-iv). Fragment A:B intensity increased monotonically with gel stiffness rather size matches predictions for MMP2 digestion (Song et al., than exhibiting an optimum (Figures 6D-i and S6G-ii). LMNA’s in- 2012) but is likely a transient intermediate that is further crease depended on myosin-II, as did cell/nuclear aspect ratio degraded (Buxboim et al., 2014)(Figure 5E-v). MS of hearts (AR), spreading, and sarcomere assembly (Figures 6C, 6D-ii, treated with blebb or collagenase not only confirmed the trend and S6A,E). Monotonic increases with stiffness of ECM agree for the fragment (>6 LMNA peptides) (Figure 5F) but also showed with elevated isometric tension as documented for other cell MMP2 levels remain constant, as supported by immunoblots for types (e.g., Engler et al., 2006). Remarkably, MMP-i or MMP2-i MMP2’s activation (cleaved/pro-MMP2 ratio; Figures 5G and rescued the blebb-induced decrease in lamin-A:B at the sin-gle- S5B). Anti-MMP2 (for both pro- and active forms) was mostly cell level (Figure 6D-i) as seen for intact embryonic hearts (Figure nucleoplasmic in isolated CMs with localization unaffected by 5D-i) despite no significant effects on cell morphology or actomyosin inhibition (Figure 5H). Nucleoplasmic MMP2 in striation (Figures 6C and 6D-ii).

(D) (i) Lamin-A:B increases with gel stiffness except with blebb (n > 50 cells per grp). Effects are rescued by MMP-i’s on rigid plastic. (ii) Cell and nuclear AR (inset) increase as CMs polarize on stiffer gels. Blebb reduces cell/nuclear AR, but MMP-i shows no affect (n > 47 cells per cond.; all error bars indicate ± SEM). (E) (i) LMNA is more nucleoplasmic in CMs on soft gels (0.3 kPa) than stiff (40 kPa). Blebb gives higher nucleoplasmic signal. (ii) Histograms of phosphorylation (pSer/LMNA) for pSer22 and pSer390. Signals for both sites are higher for soft gels and stiff gels + blebb. pSer/LMNA for rare mitotic CMs (yellow) are typically 10- to 20-fold higher than interphase CMs (cyan arrows). (iii) pSer/LMNA ratios for pSer22 and 390 (n > 11 nuclei per cond.). (F) (i) Phospho-mimetic mutant GFP-S22E (± Trx-i, 3 h) probed with anti-LMNA shows low LMNA versus GFP-S22A cells. Stable lines were made within a stable shLMNA line to minimize both endogenous LMNA and overexpression artifacts. Low-MW fragments (green arrows) are not detected in GFP-S22A cells. (ii and iii) Basal DNA damage and (iv) % 4 N cells are highest in GFP-S22E cells, but blebb minimizes differences (n > 93 nuclei per cond.; t test *p < 0.05, ** < 0.01, *** < 0.001; irrelevant blot lanes have been removed and is indicated by a blank space).

930 Developmental Cell 49, 920–935, June 17, 2019 Since myosin-II-dependent increases in lamin-A:B versus gel thus mechano-protects DNA by retaining repair factors in the nu- stiffness for isolated CMs (Figure 6D-i) align with lamin-A:B’s in- cleus (Figures 3A–3D), which thereby prevents excessive DNA crease with heart stiffness and contractility during development damage in stiff microenvironments and/or under high actomy- (Figures 5B and 5C), LMNA phosphorylation was re-examined osin stress (Figure 7A). Rapid change in LMNA protein indepen- (per Figures 5D and S5A). IF with anti-pSer22 and pSer390 dent of any transcription or translation (Trx-i) rules out many proved consistent with tension-suppressed LMNA phosphoryla- possible contributing pathways to the equally rapid DNA dam- tion and turnover: cells on soft gels showed high nucleoplasmic age response. Such conclusions about causality are otherwise phospho-signal as did cells on stiff gels treated with blebb difficult to achieve with experiments conducted over many hours (Figure 6E). The latter also showed cytoplasmic phospho- or days such as with mouse models. Results with the cardiac signal, consistent with multisite phosphorylation in interphase myosin-II specific inhibitor (MYK) in clinical trials for some car- (Kochin et al., 2014). Regardless, LMNA was similarly low and diomyopathies (National Institutes of Health, 2016a decreased at the nuclear envelope (Figure 6E-i). Phospho-signal [NCT02842242]) further suggest an unconventional method to normalized to total LMNA (pSer per LMNA) was also 2- to 3-fold attenuate DNA damage in the heart (Figures 1C and 1D). higher in CMs on soft gels than in stiff gels (Figures 6E-ii and 6E- Loss of DNA repair factors could compromise genome integ- iii). blebb-treated cells on stiff gels again showed increased rity, but enhanced entry of cytoplasmic nucleases (Maciejowski pSer/LMNA for both phospho-sites. Signal in interphase cells et al., 2015) would seem unlikely to generate gH2AX foci distrib- was, however, >10-fold lower than in rare mitotic CMs (Fig- uted throughout the nucleoplasm (Figures 3E-ii, 4A-i). ure 6E-ii, inset: purple arrow). Studies of stable phospho- Indeed, cytoplasmic proteins that bind DNA strongly (e.g., mimetic mutants (GFP-S22A and GFP-S22E) conformed to the cGAS) are restricted to rupture sites. Rupture results in ‘‘global’’ model, with higher nucleoplasmic signal but lower overall levels mis-localization of multiple DNA repair factors in several path- for S22E (Figures 6F-i and S7A). Inhibition of translation for 3 h ways (Figure S2E-i) (Xia et al., 2018; Irianto et al., 2017), (Trx-i) showed more degradation only for S22E (Figures 6F-i and repair factor depletion causes excess DNA damage and S7B), and MMP2-i treatment increased S22E intensity (Fig- before, during, and after transiently induced damage (by 1 h ure 7C) consistent with inhibition of phospho-LMNA turnover. etoposide; Figure 4B). S22E-phosphomimetic cells also exhibited higher DNA damage Remarkably, MMPs that degrade and remodel collagen-I (Figures 6F-ii and 6F-iii) and more cells in late cell cycle (4 N; Fig- matrix within hours or less are found here to directly regulate ure 6F-iv) that were more suppressed by acute blebb than S22A LMNA of the ‘‘nuclear matrix’’ (Figures 5D-i, 6C, and 6D), which cells. Reduction of nuclear stress by soft matrix or actomyosin indicates a surprising inside-outside symmetry (Figure 7) to the inhibition at the single-cell level thus favors LMNA interphase ‘‘use it or lose it’’ mechanism (Figures 5C and 7B) of stress-sta- phosphorylation, nucleoplasmic solubilization, and subsequent bilizing fibers. While both phospho-LMNA and MMP2 are found degradation—while also suppressing DNA damage and cell- mostly in the nucleoplasm of CMs, the specific functions of cycle arrest. MMP2 in the nucleus, the location of LMNA degradation, and its activation and localization mechanisms remain to be elucidated. DISCUSSION Nuclear MMPs are not new (Xie et al., 2017), but tension regulation of their substrates has not been described. Our LMNA defects cause disease through ‘‘cell-extrinsic mecha- studies of isolated CMs on soft or stiff gels further demon-strate nisms’’ that likely include ECM and/or cytoskeletal stress. steady-state LMNA levels are determined primarily by a basal Mosaic mice in which 50% of the cells express defective tone related to average morphologies of cells and nuclei, as LMNA maintain a normal lifespan, whereas mice with 100% opposed to dynamic contractions that intermittently strain the defective cells die within weeks of birth (de La Rosa et al., nucleus (Figures 3F, 6D, and S6G). Basal and dynamic 2013). Cultures on rigid plastic of the same cells (and similar strain might contribute to a ‘‘tension-time integral’’ model cell types [Hernandez et al., 2010]) exhibit premature senes- that seems predictive for hypertrophic versus dilated cardiomy- cence and apoptosis, as is common with excess DNA damage, opathy disease fates (Davis et al., 2016). Roles for MMPs in but growth and viability are surprisingly rescued upon culture such diseases and compensatory changes in LMNA could on almost any type of ECM. Soft matrix certainly reduces emerge. cytoskeletal stress and suppresses nuclear rupture and DNA Reasons why cells do not simply maintain high LMNA levels at damage in CMs with low LMNA (Figure 3E). The findings all times in all tissues could be physical as well as biochemical. are further consistent with the observation that laminopathies An overly rigid nucleus might disrupt cytoskeletal assembly and spare soft tissues such as brain, independent of lineage function. Also, LMNA fine tunes differentiation and matura-tion or developmental origin, but generally affect stiff and mechan- by regulating many cytoskeletal genes via the MKL1/SRF ically stressed adult tissues including muscle or bone (Cho pathway and by interacting with regulatory factors at the nuclear et al., 2018). periphery. Nonetheless, accumulation of LMNA in stiff tissues of Early embryos are soft, with minimal ECM, but as tissues form the embryo begins earlier than reported (Stewart and Burke, and sustain higher physical stresses, collagen-I accumulates, 1987) and tracks nonlinearly what eventually becomes the most and stiffening of the developing tissue minimizes the strain on abundant protein in adult animals, collagen-I (Figure 5C). resident cells. However, increasing stiffness and stress necessi- Collagenase softens tissue and decreases LMNA consistent tate an adaptive mechanism to protect against nuclear stress, with scaling trends, and the scaling LMNA collagen-I0.3 in em- and accumulation of mechanosensitive LMNA fulfills this role— bryos matches meta-analyses of all available transcriptomes unless actomyosin stress is inhibited (Figures 1A–1C). LMNA analyzed for diverse normal and diseased adult and developing

Developmental Cell 49, 920–935, June 17, 2019 931 A

‘Use it or Lose it’ model

Figure 7. LMNA Mechanosensing Based on a ‘‘Use It or Lose It’’ Mechanism for Tension-Inhibited Turnover that Also Applies to Collagen-I (A) Summary sketch. (B) (i) Gene circuit model adapted from Dingal et al. (Dingal and Discher, 2014), with (ii) governing equations. Squares, genes; circles, protein. LMNA and myosin-II protein (l and m) are weak regulators of the Serum Response Factor (SRF) pathway. LMNA upregulates its own transcription (via RARg). LMNA and collagen-I protein turnover with x and z sensitivity of degradation to myosin tension. Myosin-II protein turnover depends on matrix elasticity E, which scales with collagen-I (Figure 4E).

hearts across species (Cho et al., 2017)(Fig. S7D). The scaling from Swift et al., 2013; Ro¨ ber et al., 1989). Mechano-coupled is consistent with that across proteomes of diverse chick embryo accumulation of LMNA and collagens that begin in early devel- tissues at E18 (Fig. S7E), as well as adult mouse tissues (a = 0.4) opment could thus persist well into maturation and perhaps (Swift et al., 2013), and underscores a universality of normal even to disease and aging of adult tissues (Figure 7G). Thus, me- regulation. Even the tissue-dependent timing of LMNA detection chano-protection of the genome by LMNA likely plays a critical (Solovei et al., 2013; Ro¨ ber et al., 1989; Lehner et al., 1987) cor- role not only in embryonic development but also in a broad range relates well with stiffness of a tissue in adults (Fig. S7F; adapted of adult diseases.

932 Developmental Cell 49, 920–935, June 17, 2019 STAR+METHODS Baghirova, S., Hughes, B.G., Poirier, M., Kondo, M.Y., and Schulz, R. (2016). Nuclear matrix metalloproteinase-2 in the cardiomyocyte and the ischemic-re- Detailed methods are provided in the online version of this paper perfused heart. J. Mol. Cell. Cardiol. 94, 153–161. and include the following: Bertacchini, J., Beretti, F., Cenni, V., Guida, M., Gibellini, F., Mediani, L., Marin, O., Maraldi, N.M., de Pol, A., Lattanzi, G., et al. (2013). The protein kinase Akt/ d KEY RESOURCES TABLE PKB regulates both prelamin A degradation and Lmna gene expression. d CONTACT FOR REAGENT AND RESOURCE SHARING FASEB J. 27, 2145–2155. d EXPERIMENTAL MODEL AND SUBJECT DETAILS Bollen, I.A.E., Schuldt, M., Harakalova, M., Vink, A., Asselbergs, F.W., Pinto, € B Embryonic Chick Hearts and Cardiomyocytes J.R., Kruger, M., Kuster, D.W.D., and van der Velden, J. (2017). Genotype-specific pathogenic effects in human dilated cardiomyopathy. J. Physiol. (Lond.) B Induced Pluripotent Stem Cell-Derived Cardiomyo- 595, 4677–4693. cytes (iPS-CMs) Burke, B., and Stewart, C.L. (2013). The nuclear lamins: flexibility in function. B Cell Lines Nat. Rev. Mol. Cell Biol. 14, 13–24. d METHOD DETAILS Buxboim, A., Swift, J., Irianto, J., Spinler, K.R., Dingal, P.C., Athirasala, A., B Mass Spectrometry (LC-MS/MS) of Whole Heart Kao, Y.R., Cho, S., Harada, T., Shin, J.W., et al. (2014). Matrix elasticity regu- Lysates lates lamin-A, C phosphorylation and turnover with feedback to actomyosin. B Immunoblotting Curr. Biol. 24, 1909–1917. B Ex Vivo Drug Perturbations of Embryonic Hearts Chang, A.C.Y., Chang, A.C.H., Kirillova, A., Sasagawa, K., Su, W., Weber, G., B Morpholino Knockdown in Intact Hearts Lin, J., Termglinchan, V., Karakikes, I., Seeger, T., et al. (2018). Telomere short- B Alkaline Comet Assay ening is a hallmark of genetic cardiomyopathies. Proc. Natl. Acad. Sci. USA B Immunofluorescence Imaging 115, 9276–9281. B siRNA Knockdown and GFP-Repair Factor Rescue Cho, S., Abbas, A., Irianto, J., Ivanovska, I.L., Xia, Y., Tewari, M., and Discher, B mCherry D450A FOK1 TRF1 Expression D.E. (2018). Progerin phosphorylation in interphase is lower and less mecha- B Quantitative Fluorescence In Situ Hybridization and nosensitive than lamin-A, C in iPS-derived mesenchymal stem cells. Nucleus 9, 230–245. Telomere Analysis Cho, S., Irianto, J., and Discher, D.E. (2017). Mechanosensing by the nucleus: d QUANTIFICATION AND STATISTICAL ANALYSIS From pathways to scaling relationships. J. Cell Biol. 216, 305–315. B Quantification of Beating Strain Dahl, K.N., Ribeiro, A.J., and Lammerding, J. (2008). Nuclear shape, me- B Statistics chanics, and mechanotransduction. Circ. Res. 102, 1307–1318. Davis, J., Davis, L.C., Correll, R.N., Makarewich, C.A., Schwanekamp, J.A., SUPPLEMENTAL INFORMATION Moussavi-Harami, F., Wang, D., York, A.J., Wu, H., Houser, S.R., et al. (2016). A tension-based model distinguishes hypertrophic versus dilated car- Supplemental Information can be found online at https://doi.org/10.1016/j. diomyopathy. Cell 165, 1147–1159. devcel.2019.04.020. de La Rosa, J., Freije, J.M., Cabanillas, R., Osorio, F.G., Fraga, M.F., Ferna´ ndez-Garcıá, M.S., Rad, R., Fanjul, V., Ugalde, A.P., Liang, Q., et al. ACKNOWLEDGMENTS (2013). Prelamin A causes progeria through cell-extrinsic mechanisms and prevents cancer invasion. Nat. Commun. 4, 2268. This work was supported by the National Institutes of Health/National Heart Dingal, P.C., and Discher, D.E. (2014). Systems mechanobiology: tension-in- Lung and Blood Institute awards R01 HL124106 and R21 HL128187, National hibited protein turnover is sufficient to physically control gene circuits. Cancer Institute PSOC award U54 CA193417, the US-Israel Binational Sci- Biophys. J. 107, 2734–2743. ence Foundation, Charles Kaufman Foundation award KA2015-79197, and Dupont, S., Morsut, L., Aragona, M., Enzo, E., Giulitti, S., Cordenonsi, M., National Science Foundation grant agreement CMMI 15-48571. Zanconato, F., Le Digabel, J., Forcato, M., Bicciato, S., et al. (2011). Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183. AUTHOR CONTRIBUTIONS Engler, A.J., Carag-Krieger, C., Johnson, C.P., Raab, M., Tang, H.Y., Speicher, D.W., Sanger, J.W., Sanger, J.M., and Discher, D.E. (2008). Embryonic cardi- Conceptualization, S.C. and D.E.D.; Investigation, S.C., M.V., A.A., S.M., K.V., omyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity in- I.I., J.I., and M.T.; Validation, Y.X. and K.Z.; Formal Analysis, S.C. and D.E.D.; hibits beating. J. Cell Sci. 121, 3794–3802. Resources, E.T., F.M., H.T., R.G., and B.P.; Writing, S.C. and D.E.D.; Supervi- Engler, A.J., Sen, S., Sweeney, H.L., and Discher, D.E. (2006). Matrix elasticity sion, D.E.D.; Funding Acquisition, D.E.D. directs stem cell lineage specification. Cell 126, 677–689. Fatkin, D., MacRae, C., Sasaki, T., Wolff, M.R., Porcu, M., Frenneaux, M., DECLARATION OF INTERESTS Atherton, J., Vidaillet, H.J., Jr., Spudich, S., De Girolami, U., et al. (1999). The authors declare no competing interests. Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease. N. Engl. J. Med. 341, 1715–1724. Received: November 20, 2018 Revised: February 20, 2019 Geiger, B., Spatz, J.P., and Bershadsky, A.D. (2009). Environmental sensing Accepted: April 16, 2019 through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33. Published: May 16, 2019 Graziano, S., Kreienkamp, R., Coll-Bonfill, N., and Gonzalo, S. (2018). Causes and consequences of genomic instability in laminopathies: replication stress REFERENCES and interferon response. Nucleus 9, 258–275. Green, E.M., Wakimoto, H., Anderson, R.L., Evanchik, M.J., Gorham, J.M., Aix, E., Gutie´ rrez-Gutie´ rrez, O´ ., Sa´ nchez-Ferrer, C., Aguado, T., and Flores, I. Harrison, B.C., Henze, M., Kawas, R., Oslob, J.D., Rodriguez, H.M., et al. (2016). Postnatal telomere dysfunction induces cardiomyocyte cell-cycle ar- (2016). A small-molecule inhibitor of sarcomere contractility suppresses hy- rest through p21 activation. J. Cell Biol. 213, 571–583. pertrophic cardiomyopathy in mice. Science 351, 617–621.

Developmental Cell 49, 920–935, June 17, 2019 933 Gruenbaum, Y., Margalit, A., Goldman, R.D., Shumaker, D.K., and Wilson, K.L. Li, F., Wang, X., Capasso, J.M., and Gerdes, A.M. (1996). Rapid transition of (2005). The nuclear lamina comes of age. Nat. Rev. Mol. Cell Biol. 6, 21–31. cardiac myocytes from hyperplasia to hypertrophy during postnatal develop- Guilluy, C., Osborne, L.D., Van Landeghem, L., Sharek, L., Superfine, R., ment. J. Mol. Cell. Cardiol. 28, 1737–1746. Garcia-Mata, R., and Burridge, K. (2014). Isolated nuclei adapt to force and Li, H., Vogel, H., Holcomb, V.B., Gu, Y., and Hasty, P. (2007). Deletion of Ku70, reveal a mechanotransduction pathway in the nucleus. Nat. Cell Biol. 16, Ku80, or both causes early aging without substantially increased cancer. Mol. 376–381. Cell. Biol. 27, 8205–8214. Harr, J.C., Luperchio, T.R., Wong, X., Cohen, E., Wheelan, S.J., and Reddy, Liu, B., Wang, J., Chan, K.M., Tjia, W.M., Deng, W., Guan, X., Huang, J.D., Li, K.L. (2015). Directed targeting of chromatin to the nuclear lamina is mediated K.M., Chau, P.Y., Chen, D.J., et al. (2005). Genomic instability in laminopathy- by chromatin state and A-type lamins. J. Cell Biol. 208, 33–52. based premature aging. Nat. Med. 11, 780–785. Hernandez, L., Roux, K.J., Wong, E.S., Mounkes, L.C., Mutalif, R., Maciejowski, J., Li, Y., Bosco, N., Campbell, P.J., and de Lange, T. (2015). Navasankari, R., Rai, B., Cool, S., Jeong, J.W., Wang, H., et al. (2010). Chromothripsis and Kataegis induced by telomere crisis. Cell 163, 1641–1654. Functional coupling between the extracellular matrix and nuclear lamina by Majkut, S., Idema, T., Swift, J., Krieger, C., Liu, A., and Discher, D.E. (2013). Wnt signaling in progeria. Dev. Cell 19, 413–425. Heart-specific stiffening in early embryos parallels matrix and myosin expres- Higo, T., Naito, A.T., Sumida, T., Shibamoto, M., Okada, K., Nomura, S., sion to optimize beating. Curr. Biol. 23, 2434–2439. Nakagawa, A., Yamaguchi, T., Sakai, T., Hashimoto, A., et al. (2017). DNA sin- Naeem, A.S., Zhu, Y., Di, W.L., Marmiroli, S., and O’Shaughnessy, R.F. (2015). gle-strand break-induced DNA damage response causes heart failure. Nat. AKT1-mediated Lamin A/C degradation is required for nuclear degradation Commun. 8, 15104. and normal epidermal terminal differentiation. Cell Death Differ. 22, Huang, D.L., Bax, N.A., Buckley, C.D., Weis, W.I., and Dunn, A.R. (2017). 2123–2132. Vinculin forms a directionally asymmetric catch bond with F-actin. Science National Institutes of Health. (2016a). NCT02842242 A Phase 2 open-label pilot 357, 703–706. study evaluating MYK-461 in subjects with symptomatic hypertrophic cardio- Irianto, J., Xia, Y., Pfeifer, C.R., Athirasala, A., Ji, J., Alvey, C., Tewari, M., myopathy and left ventricular outflow tract obstruction. https://ClinicalTrials. Bennett, R.R., Harding, S.M., Liu, A.J., et al. (2017). DNA damage follows gov/show/NCT02842242. repair factor depletion and portends genome variation in cancer cells after National Institutes of Health. (2016b). NCT02929329 Registrational study with pore migration. Curr. Biol. 27, 210–223. omecamtiv mecarbil/AMG 423 to treat chronic heart failure with reduced ejec- Jacot, J.G., McCulloch, A.D., and Omens, J.H. (2008). Substrate stiffness af- tion fraction. https://ClinicalTrials.gov/show/NCT02929329. fects the functional maturation of neonatal rat ventricular myocytes. Oh, H., Wang, S.C., Prahash, A., Sano, M., Moravec, C.S., Taffet, G.E., Biophys. J. 95, 3479–3487. Michael, L.H., Youker, K.A., Entman, M.L., and Schneider, M.D. (2003). Kanellos, G., Zhou, J., Patel, H., Ridgway, R.A., Huels, D., Gurniak, C.B., Telomere attrition and Chk2 activation in human heart failure. Proc. Natl. Sandilands, E., Carragher, N.O., Sansom, O.J., Witke, W., et al. (2015). ADF Acad. Sci. USA 100, 5378–5383. and Cofilin1 control actin stress fibers, nuclear integrity, and cell survival. Osmanagic-Myers, S., Dechat, T., and Foisner, R. (2015). Lamins at the cross- Cell Rep. 13, 1949–1964. roads of mechanosignaling. Genes Dev. 29, 225–237. Katz, Z.B., Wells, A.L., Park, H.Y., Wu, B., Shenoy, S.M., and Singer, R.H. (2012). Beta-actin mRNA compartmentalization enhances focal adhesion sta- Pajerowski, J.D., Dahl, K.N., Zhong, F.L., Sammak, P.J., and Discher, D.E. bility and directs cell migration. Genes Dev. 26, 1885–1890. (2007). Physical plasticity of the nucleus in stem cell differentiation. Proc. Natl. Acad. Sci. USA 104, 15619–15624. Kochin, V., Shimi, T., Torvaldson, E., Adam, S.A., Goldman, A., Pack, C.G., Melo-Cardenas, J., Imanishi, S.Y., Goldman, R.D., and Eriksson, J.E. (2014). Paszek, M.J., Zahir, N., Johnson, K.R., Lakins, J.N., Rozenberg, G.I., Gefen, Interphase phosphorylation of lamin A. J. Cell Sci. 127, 2683–2696. A., Reinhart-King, C.A., Margulies, S.S., Dembo, M., Boettiger, D., et al. (2005). Tensional homeostasis and the malignant phenotype. Cancer Cell 8, Kubben, N., Voncken, J.W., Konings, G., van Weeghel, M., van den 241–254. Hoogenhof, M.M., Gijbels, M., van Erk, A., Schoonderwoerd, K., van den Bosch, B., Dahlmans, V., et al. (2011). Post-natal myogenic and adipogenic Prudova, A., auf dem Keller, U., Butler, G.S., and Overall, C.M. (2010). developmental: defects and metabolic impairment upon loss of A-type lamins. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes Nucleus 2, 195–207. by iTRAQ-TAILS quantitative proteomics. Mol. Cell Proteomics 9, 894–911. Kwan, J.A., Schulze, C.J., Wang, W., Leon, H., Sariahmetoglu, M., Sung, M., Puente, B.N., Kimura, W., Muralidhar, S.A., Moon, J., Amatruda, J.F., Phelps, Sawicka, J., Sims, D.E., Sawicki, G., and Schulz, R. (2004). Matrix metallopro- K.L., Grinsfelder, D., Rothermel, B.A., Chen, R., Garcia, J.A., et al. (2014). The teinase-2 (MMP-2) is present in the nucleus of cardiac myocytes and is oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro. FASEB J. through DNA damage response. Cell 157, 565–579. 18, 690–692. Raab, M., Gentili, M., de Belly, H., Thiam, H.R., Vargas, P., Jimenez, A.J., Lammerding, J., Fong, L.G., Ji, J.Y., Reue, K., Stewart, C.L., Young, S.G., and Lautenschlaeger, F., Voituriez, R., Lennon-Dume´ nil, A.M., Manel, N., et al. Lee, R.T. (2006). Lamins A and C but not lamin B1 regulate nuclear mechanics. (2016). ESCRT III repairs nuclear envelope ruptures during cell migration to J. Biol. Chem. 281, 25768–25780. limit DNA damage and cell death. Science 352, 359–362. Larrieu, D., Britton, S., Demir, M., Rodriguez, R., and Jackson, S.P. (2014). Ribeiro, A.J., Ang, Y.S., Fu, J.D., Rivas, R.N., Mohamed, T.M., Higgs, G.C., Chemical inhibition of NAT10 corrects defects of laminopathic cells. Science Srivastava, D., and Pruitt, B.L. (2015). Contractility of single cardiomyocytes 344, 527–532. differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness. Proc. Natl. Acad. Sci. USA 112, 12705–12710. Larrieu, D., Vire´ , E., Robson, S., Breusegem, S.Y., Kouzarides, T., and Jackson, S.P. (2018). Inhibition of the acetyltransferase NAT10 normalizes pro- Ro¨ ber, R.A., Weber, K., and Osborn, M. (1989). Differential timing of nuclear geric and aging cells by rebalancing the Transportin-1 nuclear import pathway. lamin A/C expression in the various organs of the mouse embryo and the Sci. Signal. 11, eaar5401. young animal: a developmental study. Development 105, 365–378. Lee, Y.K., Lau, Y.M., Cai, Z.J., Lai, W.H., Wong, L.Y., Tse, H.F., Ng, K.M., and Robison, P., Caporizzo, M.A., Ahmadzadeh, H., Bogush, A.I., Chen, C.Y., Siu, C.W. (2017). Modeling treatment response for lamin A/C related dilated Margulies, K.B., Shenoy, V.B., and Prosser, B.L. (2016). Detyrosinated micro- cardiomyopathy in human induced pluripotent stem cells. J. Am. Heart tubules buckle and bear load in contracting cardiomyocytes. Science 352, Assoc. 6, e005677. aaf0659. Lehner, C.F., Stick, R., Eppenberger, H.M., and Nigg, E.A. (1987). Differential Sawada, Y., Tamada, M., Dubin-Thaler, B.J., Cherniavskaya, O., Sakai, R., expression of nuclear lamin proteins during chicken development. J. Cell Biol. Tanaka, S., and Sheetz, M.P. (2006). Force sensing by mechanical extension 105, 577–587. of the Src family kinase substrate p130Cas. Cell 127, 1015–1026.

934 Developmental Cell 49, 920–935, June 17, 2019 Sharifi-Sanjani, M., Oyster, N.M., Tichy, E.D., Bedi, K.C., Jr., Harel, O., Taber, L.A., Sun, H., Clark, E.B., and Keller, B.B. (1994). Epicardial strains in Margulies, K.B., and Mourkioti, F. (2017). Cardiomyocyte-specific telomere embryonic chick ventricle at stages 16 through 24. Circ. Res. 75, 896–903. shortening is a distinct signature of heart failure in humans. J. Am. Heart Takaki, T., Montagner, M., Serres, M.P., Le Berre, M., Russell, M., Collinson, Assoc. 6. L., Szuhai, K., Howell, M., Boulton, S.J., Sahai, E., et al. (2017). Actomyosin Shimi, T., Kittisopikul, M., Tran, J., Goldman, A.E., Adam, S.A., Zheng, Y., drives cancer cell nuclear dysmorphia and threatens genome stability. Nat. Jaqaman, K., and Goldman, R.D. (2015). Structural organization of nuclear Commun. 8, 16013. lamins A, C, B1, and B2 revealed by superresolution microscopy. Mol. Biol. Tichy, E.D., Sidibe, D.K., Tierney, M.T., Stec, M.J., Sharifi-Sanjani, M., Cell 26, 4075–4086. Hosalkar, H., Mubarak, S., Johnson, F.B., Sacco, A., and Mourkioti, F. Shoulders, M.D., and Raines, R.T. (2009). Collagen structure and stability. (2017). Single stem cell imaging and analysis reveals telomere length differ- Annu. Rev. Biochem. 78, 929–958. ences in diseased human and mouse skeletal muscles. Stem Cell Reports 9, Shukla, P.C., Singh, K.K., Quan, A., Al-Omran, M., Teoh, H., Lovren, F., Cao, 1328–1341. L., Rovira, I.I., Pan, Y., Brezden-Masley, C., et al. (2011). BRCA1 is an essential Turgay, Y., Eibauer, M., Goldman, A.E., Shimi, T., Khayat, M., Ben-Harush, K., regulator of heart function and survival following myocardial infarction. Nat. Dubrovsky-Gaupp, A., Sapra, K.T., Goldman, R.D., and Medalia, O. (2017). Commun. 2,593. The molecular architecture of lamins in somatic cells. Nature 543, 261–264. Singh, M., Hunt, C.R., Pandita, R.K., Kumar, R., Yang, C.R., Horikoshi, N., Ulrich, T.A., de Juan Pardo, E.M., and Kumar, S. (2009). The mechanical rigid- Bachoo, R., Serag, S., Story, M.D., Shay, J.W., et al. (2013). Lamin A/C deple- ity of the extracellular matrix regulates the structure, motility, and proliferation tion enhances DNA damage-induced stalled replication fork arrest. Mol. Cell. of glioma cells. Cancer Res. 69, 4167–4174. Biol. 33, 1210–1222. Wiggan, O., Schroder, B., Krapf, D., Bamburg, J.R., and DeLuca, J.G. (2017). Siu, C.W., Lee, Y.K., Ho, J.C., Lai, W.H., Chan, Y.C., Ng, K.M., Wong, L.Y., Au, Cofilin regulates nuclear architecture through a myosin-II dependent mecha- K.W., Lau, Y.M., Zhang, J., et al. (2012). Modeling of lamin A/C mutation pre- notransduction module. Sci. Rep. 7, 40953. mature cardiac aging using patient-specific induced pluripotent stem cells. Aging 4, 803–822. Woessner, J.F., Jr., Bashey, R.I., and Boucek, R.J. (1967). Collagen development in heart and skin of the chick embryo. Biochim. biophys. Acta 140, Solovei, I., Wang, A.S., Thanisch, K., Schmidt, C.S., Krebs, S., Zwerger, M., 329–338. Cohen, T.V., Devys, D., Foisner, R., Peichl, L., et al. (2013). LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate dif- Worman, H.J., and Bonne, G. (2007). ‘‘Laminopathies’’: a wide spectrum of hu- ferentiation. Cell 152, 584–598. man diseases. Exp. Cell Res. 313, 2121–2133. Song, J., Tan, H., Perry, A.J., Akutsu, T., Webb, G.I., Whisstock, J.C., and Pike, Xia, Y., Ivanovska, I.L., Zhu, K., Smith, L., Irianto, J., Pfeifer, C.R., Alvey, C.M., R.N. (2012). PROSPER: an integrated feature-based tool for predicting prote- Ji, J., Liu, D., Cho, S., et al. (2018). Nuclear rupture at sites of high curvature ase substrate cleavage sites. PLoS One 7, e50300. compromises retention of DNA repair factors. J. Cell Biol. 217, 3796–3808. Stewart, C., and Burke, B. (1987). Teratocarcinoma stem cells and early mouse Xie, Y., Mustafa, A., Yerzhan, A., Merzhakupova, D., Yerlan, P., N Orakov, A., embryos contain only a single major lamin polypeptide closely resembling Wang, X., Huang, Y., and Miao, L. (2017). Nuclear matrix metalloproteinases: lamin B. Cell 51, 383–392. functions resemble the evolution from the intracellular to the extracellular Swift, J., Ivanovska, I.L., Buxboim, A., Harada, T., Dingal, P.C., Pinter, J., compartment. Cell Death Discov. 3, 17036. Pajerowski, J.D., Spinler, K.R., Shin, J.W., Tewari, M., et al. (2013). Nuclear Zaidel-Bar, R., Milo, R., Kam, Z., and Geiger, B. (2007). A paxillin tyrosine lamin-A scales with tissue stiffness and enhances matrix-directed differentia- phosphorylation switch regulates the assembly and form of cell-matrix adhe- tion. Science 341, 1240104. sions. J. Cell Sci. 120, 137–148.

Developmental Cell 49, 920–935, June 17, 2019 935 STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-lamin A/C (4C11) Cell Signaling Technology Cat# 4777s; RRID: AB_10545756 Mouse monoclonal antiphospho-Histone H2A.X EMD Millipore Cat# 05-636; RRID: AB_2755003 (Ser139), clone JBW301 Rabbit polyclonal anti-HSP90 Abcam Cat# ab13495; RRID: AB_1269122 Rabbit polyclonal anti-lamin B1 Abcam Cat# ab16048; RRID: AB_443298 Mouse monoclonal anti-b-actin (C4) Santa Cruz Biotechnology Cat# sc-47778; RRID: AB_2714189 Goat polyclonal anti-lamin-B (M-20) Santa Cruz Biotechnology Cat# sc-6217; RRID: AB_648158 Rabbit polyclonal antiphospho-lamin A/C (Ser22) Cell Signaling Technology Cat# 2026; RRID: AB_2136155 Rabbit polyclonal antiphospho-lamin A/C (Ser390) EMD Millipore N/A (custom) Biological Samples Human induced pluripotent stem cell-derived Stanford Cardiovascular Institute http://med.stanford.edu/scvibiobank.html cardiomyocytes (iPS-CMs) (SCVI) Biobank Chemicals, Peptides, and Recombinant Proteins Blebbistatin EMD Millipore #203390 Omecamtiv mecarbil Selleckchem S2623 MYK (MYK-581) MyoKardia N/A (gift) Collagenase Sigma-Aldrich C7657 Transglutaminase Sigma-Aldrich T5398 Ivermectin (Imp-i’) Sigma-Aldrich I8898 All-trans-retinoic acid (RA) Fisher Scientiﬁc AC207341000 Retinoic acid receptor antagonist (AGN193109) Santa Cruz Biotechnology sc-217589 GM6001 (pan-MMP inhibitor, ‘MMP-i’) EMD Millipore CC1010 ARP-100 (MMP2 inhibitor, ‘MMP2-i’) Santa Cruz Biotechnology sc-203522 Cycloheximide (CHX, ‘Trx-i’) Sigma-Aldrich C7698 PNA Probe (TelC-Cy5) PNA bio # F1003 Critical Commercial Assays Comet Assay Kit Cell Biolabs STA-350 Experimental Models: Cell Lines A549 lung carcinoma cell line ATCC CCL-185 U2OS osteosarcoma cell line expressing Laboratory of Roger Greenberg, N/A mCherry–TRF1 Univ. of Pennsylvania Experimental Models: Organisms/Strains White Leghorn chicken eggs:Speciﬁc Pathogen Charles River Laboratories Premium #10100326 Free (SPF) fertilized Oligonucleotides

Vivo-Morpholino against chick LMNA (MOLMNA): Gene Tools N/A 50-TTGGCATTCCTGCAACGCCGC-30 0 5 mismatch control Vivo-Morpholino (MOCtrl): Gene Tools N/A 50-TTCGCATTGCTGGAACGGCCC-30 Software and Algorithms MaxQuant Version 1.5.3.8 Max Planck Institute of Biochemistry http://www.coxdocs.org/doku.php?id= maxquant:common:download_ and_installation ImageJ NIH https://imagej.nih.gov/ij/ Telometer Johns Hopkins School of Medicine http://demarzolab.pathology.jhmi.edu/ telometer/index.html e1 Developmental Cell 49, 920–935.e1–e5, June 17, 2019 CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the lead Contact, Dennis E. Discher ([email protected]).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Embryonic Chick Hearts and Cardiomyocytes White Leghorn chicken eggs (Charles River Laboratories; specific pathogen free (SPF) Fertilized eggs, Premium #10100326) were used to extract embryonic hearts. SPF chicken eggs from Charles River are produced using filtered-air positive-pressure (FAPP) poultry housing and careful selection of layer flocks. Every flock’s SPF status is validated in compliance with USDA memorandum 800.65 and European Pharmacopoeia 5.2.2 guidelines. SPF eggs were incubated at 37 C with 5% CO2 and rotated once per day until the desired developmental stage (e.g. four days for E4; Hamburger-Hamilton stage 23–24 (HH23-24)). Embryos were extracted at room temperature (RT) by windowing eggs, carefully removing extraembryonic membranes with sterile sonicated forceps, and cutting major blood vessels to the embryonic disk tissue to free the embryo. The extracted embryo was then placed in a dish containing PBS on a 37C-heated plate, and quickly decapitated. For early E2-E5 embryos, whole heart tubes were extracted by severing the conotruncus and sino venosus. For more mature (>E5) embryos, embryonic disks were extracted by windowing the egg, cutting out the embryo with the overlying vitelline membrane intact, lifting out the embryo adherent to the vitelline membrane and placing in a dish of prewarmed PBS. Extraembryonic tissue was carefully cut away using dissection scissors and the embryo was teased away from the vitelline membrane using forceps. Whole hearts (>E5) were extracted by severing the aortic and pulmonary vessels. The pericardium was carefully sliced and teased away from the ventricle using extrafine forceps. E10 brain and liver tissue were collected from the presumptive midbrain and hepatic diverticulum, respectively. All tissues were incubated at 37C in prewarmed chick heart media (a-MEM supplemented with 10 % FBS and 1% Penn-strep, Gibco, #12571) for at least 1 hrs for stabilization, until ready for use. To isolate primary embryonic CMs from tissue, whole hearts were diced to submillimeter size and digested with trypsin/EDTA (Gibco, #25200-072). Approximately 1 ml of trypsin per E4 heart was added and incubated for 13 min at 37C with gentle shaking, then placed upright for 2 min to let large pieces of tissue settle to the bottom of the dish. The supernatant was carefully removed and replaced with an equal volume of fresh trypsin for a second round of 15-min incubation at 37C. Digestion was blocked by adding an equal volume (1:1) of chick heart media. Isolated cells were plated onto collagen-I-coated polyacrylamide (PA) gels and were allowed to adhere for >4 hrs. Spontaneously beating CMs were imaged using an Olympus I81 microscope with a 403 air objective configured for phase-contrast after 24 hrs in culture. As with intact embryonic hearts, cell area and AR were measured with time to quantify beating. All experiments were performed in accordance with the guidelines of the IACUC of University of Pennsylvania.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes (iPS-CMs) Normal human iPS cells (obtained from Dr. Joseph Wu, Stanford Cardiovascular Institute (CVI) biobank) were cultured following the protocol provided: Matrigel (BD Matrigel, hESC qualified: #354277) was suspended in cold 4C DMEM/F12 medium 1:200 dilution (DMEM/F12 medium #10-092-CM-Fisher), mixed gently, and 1 ml of this suspension was added to one 6-well plate (Corning Catalog #353046) and incubated for 1 hr at RT to allow Matrigel to coat the surface. The solution was gently aspirated and small aggregates of human iPS cells were added to each well in mTesr1 medium containing 10 mM of ROCK inhibitor (Y27632, 2HCl – 50 mg: #50-863-7- Fisher). Culture medium was replaced daily (no ROCK inhibitor) until the cells reached 85% confluency. Human induced pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (hiPS-CMs) using the ‘‘Cardiomyocytes differentiation and maintenance kit’’ from Stem Cell Technologies (#05010 & #05020). The differentiation process was followed as described by the manufacturer’s protocol. Briefly, the mTesr1 medium with ROCK inhibitor (10 mM) was replaced with differentiation medium A, cultured for 2 days (Day 0), then subsequently to medium B for 2 days (Day 2) and again switched to medium C twice (Day 4 & 6). From Day 8 onwards, maintenance medium (Day 8) was added/refreshed every 2 days until spontaneous CM beating was observed through imaging.

Cell Lines A549 (human lung adenocarcinoma) cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and U2OS cell lines were obtained from the laboratory of Roger Greenberg, University of Pennsylvania. ATCC provides cell line authentication test recommendations per Tech Bulletin number 8 (TB-0111-00-02; 2010). These include: microscopy-based cell morphology check, growth curve analysis, and mycoplasma detection (by DNA staining) which were conducted on all cell lines used in these studies. All cell lines maintained the expected morphology and standard growth rates with no mycoplasma detected. U2OS and A549 cell lines were cultured in DMEM high-glucose media and Ham’s F-12 nutrient mixture (Gibco, Life Technologies), respectively, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Sigma- Aldrich).

Developmental Cell 49, 920–935.e1–e5, June 17, 2019 e2 METHOD DETAILS

Mass Spectrometry (LC-MS/MS) of Whole Heart Lysates Mass spectrometry (LC-MS/MS, or ‘MS’) samples were prepared using procedures outlined in Swift et al.. Briefly, 1mm3 gel sections were carefully excised from SDS–PAGE gels and were washed in 50% 0.2-M ammonium bicarbonate (AB), 50% acetonitrile (ACN) solution for 30 min at 37C. The washed slices were lyophilized for >15 min, incubated with a reducing agent (20 mM TCEP in 25-mM AB solution), and alkylated (40 mM iodoacetamide (IAM) in 25-mM AB solution). The gel sections were lyophilized again before in-gel trypsinization (20 mg/mL sequencing grade modified trypsin, Promega) overnight at 37C with gentle shaking. The resulting tryptic peptides were extracted by adding 50% digest dilution buffer (60-mM AB solution with 3% formic acid) and injected into a high-pressure liquid chromatography (HPLC) system coupled to a hybrid LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray ion source. Raw data from each MS sample was processed using MaxQuant (version 1.5.3.8, Max Planck Institute of Biochemistry). MaxQuant’s built-in Label-Free Quantification (LFQ) algorithm was employed with full tryptic digestion and up to 2 missed cleavage sites. Peptides were searched against a FASTA database compiled from UniRef100 Gallus gallus (chicken; downloaded from UniProt), plus contaminants and a reverse decoy database. The software’s decoy search mode was set as ‘revert’ and a MS/MS tolerance limit of 20 ppm was used, along with a false discovery rate (FDR) of 1%. The minimum number of amino acid residues per tryptic peptide was set to 7, and MaxQuant’s ‘match between runs’ feature was used for transfer of peak identifications across samples. All other parameters were run under default settings. The output tables from MaxQuant were fed into its bioinformatics suite, Perseus (version 1.5.2.4), for protein annotation and sorting.

Immunoblotting Hearts isolated from chick embryos (E4, E6, and E10; n R 8, 4, and 2 per sample, respectively) were rinsed with prewarmed PBS, excised into submillimeter pieces, quickly suspended in ice-cold 13 NuPage LDS buffer (Invitrogen; diluted 1:4 in 13 RIPA buffer, plus 1% protease inhibitor cocktail, 1% b-mercaptoethanol), and lysed by probe-sonication on ice (10 3 3 s pulses, intermediate power setting). Samples were then heated to 80C for 10 min and centrifuged at maximum speed for 30 min at 4C. SDS-PAGE gels were loaded with 5 - 15 mL of lysate per lane (NuPage 4%-12% bis-Tris; Invitrogen). Lysates were diluted with additional 13 NuPage LDS buffer if necessary. Gel electrophoresis was run for 10 min at 100 V and 1 hrs at 160 V. Separated samples were then transferred to a polyvinylidene ﬂuoride membrane using an iBlot Gel Transfer Device (Invitrogen). The membrane was blocked with 5% nonfat dry milk in TTBS buffer (Tris-buffered saline, BioRad; with 0.1% Tween-20), washed 33 in TTBS, and incubated with primary antibodies against: LMNA (1:500, CST, #4777), gH2AX (1:1000, EMD Millipore, #05-636), HSP90 (1:1000, Abcam, #ab13495), b-actin (1:200, Santa Cruz, #sc-47778), LMNB (1:200, Santa Cruz, #sc-6217), LMNB1 (1:1000, Abcam, ab16048), LMNA pSer22 (1:500, CST, #2026), and/or LMNA pSer390 (1:500, EMD Millipore, custom-made Ab) diluted in TBS to ﬁnal concentrations generally on the order of 1 mg/ml at 4C overnight. After washing 33 with TTBS, the membrane was incubated with 1:2000 diluted secondary Ab: anti-mouse HRP-conjugated IgG (GE Healthcare, Little Chalfont, UK), at RT for R1.5 hrs. The membrane was washed 33 again with TTBS and developed with ChromoSensor (GenScript) for 3 min at RT. Immunoblot images were obtained using a HP Scanjet 4850. Densitometry was performed using ImageJ (NIH).

Ex Vivo Drug Perturbations of Embryonic Hearts For actomyosin perturbation experiments, intact E4 hearts were incubated in 25 mM blebbistatin (EMD Millipore, #203390, stock solution 50 mg/ml in DMSO), 1 mM omecamtiv mecarbil (OM, cytokinetics), or 0.3 - 1 mM MYK-581 (gift from MyoKardia, analog of MYK-461 (Green et al., 2016)) in heart medium at 37C, for the desired duration. Drug solutions were kept away from light to minimize photodeactivation. Treated hearts were compared to a control sample treated with an equal concentration of vehicle solvent DMSO in heart culture media. Drug solutions were gently washed-out with prewarmed chick heart media 33 for recovery experiments. For collagen matrix perturbations, E4 heart tissue was incubated in 0.3 - 1-mg/ml concentrations of collagenase (Sigma, #C7657) for 45 min, or 20-mg/mL transglutaminase (Sigma, T5398) for up to 3 hrs at 37C. Enzyme activity was blocked by replac- ing with chick heart media containing 5% BSA. Nuclear import/export of KU80 was inhibited using 5 mM ivermectin (Sigma I8898), 10 mM mifepristone (Sigma M8046), or45 nM leptomycin B (Sigma L2913) for 24 hrs. For transcriptional modulation of LMNA, hearts were incubated with 1 mM retinoic acid (RA, Fisher Scientiﬁc) or antagonist to retinoic acid (AGN-193109, Santa Cruz), for short (3 hrs) or long (72 hrs) treatment times. For MMP inhibition during blebbistatin treatment, hearts were incubated with 10 mM GM6001 (‘MMP-i’, EMD Millipore, #CC1010) or with 30 mM of ARP-100 (‘MMP2-i’,Santa Cruz, CAS 704888-90-4). 1 mM cycloheximide (‘Trx-I’, Sigma, C7698-1G) was added to block protein synthesis during actomyosin and/or matrix perturbations. 5 mM ivermectin (Sigma, I8898) was used to inhibit nuclear import. A minimum of 8 hearts were treated and pooled per lysate/experimental condition.

Morpholino Knockdown in Intact Hearts Vivo-Morpholinos (MO) against LMNA mRNA and the corresponding 50-mispair control were designed according to the manufac- 0 0 0 0 turer’s guidelines (Gene Tools, Inc.): 5 -TTGGCATTCCTGCAACGCCGC-3 (MOLMNA) and 5 -TTCGCATTGCTGGAACGGCCC-3 (MOCtrl). Desired concentrations (1, 2, 5, 10, 30 mM) were prepared by dilution in heart growth medium at 37 C, and the resulting solutions were added to E4 hearts for 24 hrs before analysis. e3 Developmental Cell 49, 920–935.e1–e5, June 17, 2019 Alkaline Comet Assay Alkaline comet assays was performed according to manufacturer’s protocol (Cell Biolabs). Briefly, cells were trypsinized, mixed with liquefied agarose at 37C, placed drop-wise onto the supplied glass slide, and incubated for 15 min at 4C for the agarose to gel. Lysis buffer from the kit was then added to the solidified gel and incubated for 45 min, followed by an additional 30-min incubation with alkaline solution. Electrophoresis was performed at 300 mA for 30 min followed by a 70% ethanol wash, before samples were air dried overnight. Finally, DNA dye in the kit was added to each sample for 15 minutes, followed by epifluorescence imaging as described above.

Immunofluorescence Imaging Cells were rinsed with prewarmed PBS, fixed with 4% paraformaldehyde (PFA, Fisher) for 15 min, washed 33 with PBS and permeabilized with 0.5% Triton-X (Fisher) in PBS for 10 min. Permeabilized cells were then blocked with 5% BSA in PBS for R1.5 hrs. Sam- ples were incubated overnight with primary antibodies in 0.5% BSA solution, with gentle agitation at 4C. The primary antibodies used were: LMNA (1:500, CST, #4777), LMNB (1:500, Santa Cruz, #sc-6217), LMNA pSer22 (1:500, CST, #2026), LMNA pSer390 (1:500, EMD Millipore, new custom-made Ab), a-actinin-2 (1:500, Abcam, #ab9465), gH2AX (EMD Millipore, #05-636), KU80 (1:500, Cell Signaling), 53BP1 (1:300, Novus, #NB100-304), RPA2 (1:500, Abcam). Samples were washed 33 in 0.1% BSA in PBS and incubated with the corresponding secondary antibodies at 1:500 dilution for 1.5 hrs at RT (Alexa Fluor 488, 546 and 647 nm; Invitrogen). Cells on gels or glass coverslips were mounted with mounting media (Invitrogen ProLong Gold Antifade Reagent). Images of adherent cells were taken with an Olympus IX81. All images in a given experiment were taken under the same imaging conditions and were analyzed using ImageJ (NIH). For confocal imaging of embryonic heart tissue, heart samples were rinsed with PBS, fixed with 4% paraformaldehyde (PFA, Fisher) for 45 min, washed 33 with PBS, and permeabilized with 0.5% Triton-X (Fisher) in PBS for 3 hrs. Samples were then incubated overnight with primary antibodies in 0.5% BSA solution with gentle agitation at 4C (as described above), and washed 33 in 0.1% BSA and 0.05% Triton-X in PBS. Corresponding secondary antibodies were added at 1:500 dilution for 1.5 hrs at RT. Immunostained hearts were then mounted between glass coverslips with mounting media (Invitrogen ProLong Gold Antifade Reagent). Images were taken with Leica TCS SP8 system with either a 633/1.4 NA oil-immersion or 403/1.2 NA water-immersion objective. siRNA Knockdown and GFP-Repair Factor Rescue All siRNAs used in this study were purchased from Dharmacon (ON-TARGETplus SmartPool; siBRCA1, L-003461-00; siBRCA2, L-003462-00; siKu80, L-010491-00; siRPA1, L-015749-01; siLMNA, L-004978-00 and nontargeting siRNA, D-001810-10). GFP- KU70 and GFP-KU80 were gifts from Dr. Stuart L Rulten from University of Sussex, Brighton, UK, and GFP-53BP1 was a gift from Dr. Roger Greenberg from University of Pennsylvania. Cells were plated 24 hours before transfection. Lipofectamine/nucleic- acid complexes were prepared according to the manufacturer’s instructions (Lipofectamine 2000, Invitrogen), by mixing siRNA (25 nM) or GFPs (0.2–0.5 ng/ml) with 1 mg/ml Lipofectamine 2000. Final solutions were added to cells and incubated for 3 days (for siRNAs) or 24 hours (for GFPs) in corresponding media containing 10% FBS. mCherry D450A FOK1 TRF1 Expression Death domain (DD)– estrogen receptor (ER)–mCherry–TRF1–FokI constructs were cloned and concentrated TRF1-FokI lentivirus with Polybrene (8 mg/ml) diluted in media was added to U2OS cells at a minimum titer. Doxycycline-inducible TRF1–FokI lines were generated using the Tet-On 3G system. Doxycycline was used at a concentration of 40 ng/ml for 16–24 hrs to induce expression of TRF1– D450A FokI. Shield-1 (Cheminpharma LLC) and 4-hydroxytamoxifen (4-OHT) (Sigma) were both used at a concentration of 1 mM for 2 hrs, to allow for TRF1–D450A FokI stabilization and translocation into the nucleus.

Quantitative Fluorescence In Situ Hybridization and Telomere Analysis Isolated cells were attached on slides, fixed and stained for PNA telomeric probe as we previously described in (Tichy et al., 2017). Images of mMuSCs were taken using a Nikon eclipse 90i wide-field epifluorescence microscope equipped with a Prior Proscan III motorized stage, a Photometrics Coolsnap HQ2 14-bit digital camera, and a Nikon 1003/1.40 Plan Apo VC objective. Telomeres were analyzed with the investigators blinded to genotypes and/or conditions using open-source software Telometer, as previously described. The program generates statistics on the entire region of the nucleus. Analysis includes the intensity sum of all Cy5 telomere pixels for a given nucleus (proportional to the cell’s total telomere length) and the intensity sum of all DAPI pixels for the nucleus (proportional to total cellular nuclear DNA content).

QUANTIFICATION AND STATISTICAL ANALYSIS

Quantification of Beating Strain Transfected E4 chick hearts were video-imaged using an Olympus I81 at 43 magnification, with a CCD camera. Procedures described by Taber et al. (Taber et al., 1994) were followed to calculate 2D tissue beating strain. Briefly, R3 groups of 3 cells located within 20 mm of each other were selected as fiduciary markers along the outer walls of ventricular tissue. A custom MATLAB program was written to track their displacements and compute trace of the tissue strain tensor (Taber et al., 1994). Alternatively, for higher- throughput quantitation of contractility changes (n>10 hearts per sample, e.g. following blebbistatin treatment), morphological

Developmental Cell 49, 920–935.e1–e5, June 17, 2019 e4 measurements (projected 2D area, aspect ratio (AR), circularity, perimeter, etc.) of beating whole-hearts were traced with time. Morphology measurements were normalized to baseline reference values, and peak-to-valley ‘amplitudes’ were averaged over

>5 beats to quantify tissue beating strain (e.g. DAR/ARref). A similar approach was taken to quantify nuclear deformations: the projected 2D area and aspect ratio (AR) of transfected nuclei (e.g. expressing GFP-LMNA) were traced with time, and the normalized amplitudes were measured and compared across experimental conditions.

Statistics All statistical analyses were performed and graphs were generated using GraphPad Prism 5. All error bars reflect ± SEM. Unless stated otherwise, all comparisons for groups of three or more were analyzed by one-way ANOVA followed by a Dunnett’s multiple comparison test. Pairwise sample comparisons were performed using Student’s t-test. Population distribution analyses were performed using the Kolmogorov-Smirnov test, with a = 0.05. Information regarding statistical analyses are included in the figure legends. For all figures, the p-values for statistical tests are as follows: n.s. = not significant, *p<0.05, **<0.01, ***<0.001 (or #p<0.05, ##<0.01 for multiple tests within the same dataset).

e5 Developmental Cell 49, 920–935.e1–e5, June 17, 2019 Developmental Cell, Volume 49

Supplemental Information

Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest

Sangkyun Cho, Manasvita Vashisth, Amal Abbas, Stephanie Majkut, Kenneth Vogel, Yuntao Xia, Irena L. Ivanovska, Jerome Irianto, Manorama Tewari, Kuangzheng Zhu, Elisia D. Tichy, Foteini Mourkioti, Hsin-Yao Tang, Roger A. Greenberg, Benjamin L. Prosser, and Dennis E. Discher Cho…Discher Mechanoprotecting the developing genome Feb 2019

Supplemental Information

Supplemental Figure Legends Figure S1. Reversible inhibition of contractility and transcriptional suppression of LMNA result in increased DNA damage, Related to Figures 1 & 2. (A) (i) Full-length Western blot (top) and corresponding Coomassie Brilliant Blue-stained gel (bottom) for data shown in Fig.1B&C. Key time points are highlighted in colors. (ii) Densitometry line profiles along the electrophoresis axis (vertical colored arrows) shows significant changes in LMNA and γH2AX, but not β-actin (loading control). (iii) Coomassie Brilliant Blue total intensity along each lane shows total protein content remains unchanged across conditions. All error bars indicate ±SEM. (B) (i) WB for a key time point (1h post-blebb treatment) using an alternative housekeeping protein HSP90 as a loading control (top). Densitometry line profiles (bottom) show similar ~35% reduction in LMNA but not HSP90, even in the presence of a protein synthesis inhibitor, (CHX, or ‘Trx-i’). (ii) Coomassie Brilliant Blue total intensity remains unchanged (<10%) across conditions (n=8 hearts per lysate). (C) Mass spectrometry (MS) of heart lysates detects 29 peptides unique to LMNA, and verifies that a broad range of housekeeping proteins, including LMNB1, LMNB2, GAPDH, HSP70, β-tubulin, and β-actin, remain unchanged with blebbistatin treatment (± Trx-i). n=8 hearts per lysate.

(D) (i) Quantification of tissue beating strain ΔAR/ARref and (ii) heart rate (beats / min; BPM) in blebbistatin treated hearts. Myosin-II inhibition by blebbistatin rapidly suppresses beating (<30 min), but effects are reversible such that washout of drug with culture medium (± OM) results in near full recovery by 1h. (E) (i) LMNA and γH2AX immunoblots with different doses of MO. (ii) Low dowses (<5 μm) have no

significant effect on beating strain (ΔAR/ARref) or rate (BPM), but higher doses suppress beating, indicating potential cytotoxicity (one-way ANOVA; *p<0.05, **<0.01). (F) (i) Retinoic acid (RA) and antagonist to retinoic acid (AGN) treatment in intact embryonic hearts have no observable effect on LMNA levels or DNA damage (γH2AX) at 3h. (n=6 hearts per cond.). (ii) Significant changes in LMNA and γH2AX are detectable only after 72h treatment, consistent with slow transcriptional modulation. A reduction in LMNA upon RA treatment (72h) is accompanied by an increase in DNA damage as measured by γH2AX, while AGN treatment leads to an upregulation of LMNA coupled to suppression of DNA damage (n=8 hearts per lysate, t-test; *p<0.05). (G) LMNA WB of RA/AGN treated E4 hearts, with three different loading volumes (Low, Med, High). Bottom left: LMNA vs HSP90 scatter plot (all R2 > 0.99); Right: linear fits from LMNA vs HSP90 scatter plot show slopes for AGN > Untr. > RA. (H) RA and AGN treatment does not significantly affect contractility of hearts even after 72h of treatment.

Page 1 of 5

Cho…Discher Mechanoprotecting the developing genome Feb 2019

Figure S2. Suppression of LMNA levels in intact embryonic hearts and in beating hiPS- CMs increase rupture under high stress, causing prolonged (>1h) loss of repair factors from the nucleus and accumulation of DNA damage, Related to Figure 3. (A) (i) Blebbistatin treatment and washout have no significant effect on cell death/viability, as determined by %-transfected cells with fragmented DNA. (ii) As with embryonic hearts, blebbistatin washout results in an increase in rupture with cytoplasmic mis-localization of KU80. An excess of rupture is seen with washout plus a nuclear import inhibitor (ivermectin, ‘Imp-i', which inhibits Impα/β mediated import), but not with washout plus a specific inhibitor of integrase(IN) nuclear import (mifepristone, ‘Mifepr.’) that does not target KU80’s NLS. On the other hand, washout with a nuclear export inhibitor (leptomycin B, ‘Exp-I’) greatly suppresses cytoplasmic mis-localization of KU80 (t-test *p<0.05, **<0.01). All error bars indicate ±SEM. (B) As seen with nuclei in intact embryonic hearts, ‘nuclear beating’ occurs in hiPS-CMs and can be quantified by changes in nucleus area and DNA mean intensity (condensation/de-condensation of DNA), which are inversely correlated. (C) Time-lapse images of nuclei probed with a pointed (<1 μm) Atomic Force Microscopy (AFM) tip (at ~7 nN). Nuclear rupture upon stress is evident in the rapid and stable accumulation of a cytoplasmic protein that binds DNA (GFP-cGAS). (D) (i) Nuclear/cytoplasmic KU80 IF intensity ratio decreases with siLMNA knockdown, and (ii) anti- correlates with γH2AX foci count (one-way ANOVA; ***p<0.001). (E) (i) Cytoplasmic mis-localization is not limited to KU80, but applicable to other DNA repair factors including 53BP1 and RPA2 (top: IF images, bottom: intensity profiles along white dashed line). Scale bar = 10 μm. (ii) Time-lapse images of siLMNA knockdown cells transduced with GFP-53BP1 show that nuclear rupture and cytoplasmic mis-localization occur within minutes and are maintained for at least 1h in culture indicating slow recovery. (F) Total (nuclear + cytoplasmic) KU80 abundance is unaffected by siLMNA or blebbistatin treatment. (G) Kymograph of beating hiPS-CM organoids generated by tracing length along yellow line with time. Blebbistatin treatment results in reversible inhibition of contractility.

Page 2 of 5

Cho…Discher Mechanoprotecting the developing genome Feb 2019

Figure S3. Repair factor loss perturbs cell cycle, but effects are rescued by myosin-II inhibition, Related to Figure 4. (A) (i) Histograms of normalized DNA content in hiPS-CMs after knockdown of LMNA and DNA repair factors. All repair factor knockdowns results in a higher fraction of ‘4N’ cells, with effects most severe for siCombo. (ii) Kolmogrov-Smirnov test (α = 0.05) for siRNA treated populations. (iii) γH2AX foci count inversely correlates with KU80 nuclear IF intensity at the single cell level, consistent with limited repair. All error bars indicate ±SEM. (B) DNA damage per total DNA is elevated in 4N cells after LMNA knockdown and decreased in 2N cells, based again on measurements of individual cells. (C) Left: Representative scatter plot of EdU intensity versus total DNA content used to determine cell cycle phase (G1, S, and G2). Right: siLMNA treatment increases fraction of cells in G2, but effects are rescued by blebbistatin (t-test *p<0.05, ***<0.001). (D) (i) Venn diagram of nuclear proteins pulled down by co-immunoprecipitation mass spectrometry (CoIP- MS) using anti-GFP on GFP-KU70 expressing cells. (ii) As expected, KU80 is a strong interaction partner of KU70 (as components of the KU complex), as are lamins-A and B.

Figure S4. Parallel increases in lamin-A:B, collagen matrix, and tissue stiffness fit a ‘lose it or use it’ model of tension-stabilization of fibers, Related to Figure 5. (A) LMNA MS intensity scales with heart tissue stiffness with exponent α ~ 0.75 similar to that for adult tissue proteomes (Swift, et al., 2013). All error bars indicate ±SEM. (B) Lamins-B1 & B2 remain comparatively constant throughout development and exhibit much weaker scaling (α < 0.25). (C) Heartmap of proteins detected by LC-MS/MS in the ECM, adhesion complexes, sarcomeres, and the nuclear lamina. Proteins were ranked based on the fold-change relative to the average. (D) Lamin-A:B ratio quantified by (i) WB densitometry and (ii) IF (t-test *p<0.05). (E) MS measurements of collagen-I calibrated with spike-ins of known amounts of purified collagen-I. Dividing calibrated measurements by reported myocardium volumes (Kim, et al., 2011) gives collagen- I densities far lower than those used in studies of collagen-I gels (Yang, et al., 2009), suggesting lower densities are needed for a stiffness in the kPa range as measured for heart (Fig.4C-ii). (F) Heart stiffness measurements by micropipette aspiration plotted against collagen-I MS intensity yield power-law scaling comparable to that found for diverse adult tissues (Swift, et al., 2013). (G) LMNA & B immunoblots for E10 brain and liver tissue.

Page 3 of 5

Cho…Discher Mechanoprotecting the developing genome Feb 2019

Figure S5. Acute drug perturbations in E4 heart rapidly impact LMNA levels and phosphorylation at multiple sites, but do not affect total abundance of repair factors or MMP2, Related to Figure 5. (A) Immunoblot validation of LMNA trends seen in MS profiling of drug perturbations (n>6 hearts per lysate). Immunoblots against phosphorylated Ser22 and Ser390 show that normalized phosphorylation (‘pSer22/LMNA’ and ‘Ser390/LMNA’) increases with blebbistatin-inhibition of actomyosin stress or with collagenase-softening of tissue, but decreases with CDK-i treatment (one-way ANOVA; *p<0.05, ** p<0.01, ***p<0.001). All error bars indicate ±SEM. (B) (i) RPA1 (DNA repair factor) and (ii) pro-MMP2 levels are unaffected by drug perturbations (per MS). (iii) Catalytic activation of MMP2 (‘cleaved/pro-MMP2’ fraction, as measured by immunoblot) is not affected by drug perturbations to contractility and/or collagen matrix. (C) Schematic plot of fractional abundance of intact LMNA and its phosphorylated fragments, including intermediates, upon relaxation of stress.

Figure S6. Cell-on-gel morphological trends in CM size, shape, contractility, and LMNA mirror those of in vivo hearts without affecting cell cycle/proliferation, Related to Figure 6. (A) (i) Projected area of the nucleus relative to that of the cell (‘Nucl./Cyto. Area fraction’) decreases (ii) and cells elongate (increased aspect ratio, AR), as the embryonic heart stiffens and cells undergo hypertrophic growth and spreading from E4 – E10. Isolated E4 CMs cultured on gels likewise exhibit increased cell spreading and elongation on stiffer gels (t-test *p<0.05, **<0.01, ***<0.001). All error bars indicate ±SEM. (B) Two of the most abundantly expressed α- and β-tubulin isoforms (TUBA1C, TUBB7) increase in level from E4 to E10 and the increase is accompanied by a decrease in TTLL12, which tyrosinates and de- stabilizes microtubules (Robison, et al., 2016). Trends are consistent with increased stiffness as well as with polarization/elongation of CMs during development (Fig.4D-ii, Fig.S6A). (C) Axial alignment of cells (anisotropy, quantified as 1/COV of the major axes of nuclei) increases from early (E4) to late (E11) hearts. Upper left inset: major axis angle distribution of E4 and E11 nuclei. (D) Nuclear volume (estimated by confocal Z-stack) decreases in development from E4 to E10. (E) Varying matrix stiffness alone in E4 CM cultures is sufficient to recapitulate trends in morphology and intracellular organization seen in vivo (from E4 to E11).

(F) Normalized cell and nuclear beating strains measured by (i) ‘ΔAR/ARref’ and (ii) ‘ΔArea/Arearef’, and (iii) %-beating cells all exhibit an optimum on gels mimicking the stiffness of embryonic hearts (~2 kPa). Blebbistatin treatment abolishes mechano-sensitivity. (n>10 cells/nuclei per condition).

(G) (i) Nuclear beating strain (‘Nucleus ΔAR/ARref’) correlates well with cell beating strain (‘Cell ΔAR/ARref’), (ii) but lamin-A:B ratio does not correlate with beating. Lamin-A:B instead couples to average morphology changes in spreading area and elongation that relate to basal isometric tension.

Page 4 of 5

Cho…Discher Mechanoprotecting the developing genome Feb 2019

(H) Lamin-A:B in cells on rigid plastic decreases with MYK inhibition of cardiac myosin contractility (as with blebbistatin treatment), but remains unchanged with OM treatment.

Figure S7. Phosphorylation of LMNA favors degradation by MMP2, Related to Figure 6 & 7. (A) Representative images of cells transduced with phsopho-mimetic mutants GFP-S22A and GFP-S22E, with or without protein synthesis inhibitor Trx-i. (i,ii) Line profiles across individual nuclei reveal ‘non- phosphorylatable’ GFP-S22A signal is far more enriched at the lamina than in the nucleoplasm, compared to ‘constitutively phosphorylated’ GFP-S22E (n>15 nuclei). Treatment with Trx-i does not alter nucleoplasm/lamina ratio in either mutant. (iii) Overall fluorescence intensity of GFP-S22A is ~50% higher than that of GFP-S22E. Trx-i induces a minor ~10% decrease in either case. All error bars indicate ±SEM. (B) Immunoblots with anti-lamin-A/C and anti-GFP reveal multiple low-MW degradation fragment bands (green triangles) in the GFP-S22E mutant which are absent in the S22A mutant. (ii) Line intensity profile of anti-lamin-A/C immunoblot (i, left). Low-MW degradation fragments that are present in the GFP- S22E mutant but not in the GFP-S22A mutant, are shaded in blue, with green arrows indicating distinct bands. (C) GFP fluorescence intensity of GFP-S22E expressing cells increases upon MMP2-I (ARP-100) treatment, consistent with inhibition of degradation. (D) Representative transcriptomics dataset for mouse embryonic hearts obtained from NCBI’s Gene Expression Omnibus (GEO) Database. Log-log plot shows normalized mRNA expression vs Col1a1. As expected for obligate heterotrimer subunits of collagen-I, Col1a2 correlates robustly with Col1a1,

with scaling exponent (= slope on a log-log plot), αCol1a2 ~ 1. Lmna also increases with Col1a1, although

with slightly weaker scaling αLmna ~ 0.3, indicating potential feedback to gene expression. Right: bar graph of average scaling exponents (vs Col1a1) for proteins of interest in the ECM, cytoskeleton, nuclear envelope, as well as several transcription factors implicated in mechanosensing and/or cardiac development. (E) Proteomics dataset for diverse E18 chick embryonic tissues, adapted from Uebbing et al. (Uebbing et

al., 2015). Lmna again increases with Col1a1&2, with exponent αLmna ~ 0.3. Right inset: datapoints for heart samples plotted separately reveal similar scaling. (F) Tissue-dependent timing of initial LMNA expression in early embryos correlates well with the stiffness that a given tissue eventually achieves in adult (adapted from (Swift, et al., 2013; Rober, et al., 1989)).

Page 5 of 5

Figure S1 0DVV6SHFFRQILUPV:%TXDQWLWDWLRQRI/01A & DNAGDPDJH DQGUHYHDOVKRXVHNHHSLQJSURWHLQV LQFOXGLQJ/01%’V UHPDLQXQFKDQJHGZLWKDFWRP\RVLQSHUWXUEDWLRQV

3h) W K A 5h) 5h) B C 5h) 5h) (+1h) (+ :HVWHUQEORW %OHEE 0DVV6SHF /&0606 RI(ZKROHKHDUW iiiRXW RXW i h h bb (0 bb (1h)bb (2h)bb (3h)bb (4 DMSO %OHEE 7U[L H H H H H DV DV OM (7 N'D SHSWXQLTXHWR/01$ SHq FRvHrDJH = DMSOBl (0 Bl Bl Bl Bl W W DMSO + (6h) N'D DMSO (05h) HSP90 90 80 100 SHSWVKDUHGZLWK/01% )'5 ) EDFWLQ 73

LMNA LMNA BlHbb (05h) 1 MSTPSQRRSG RGGGPSGTPL SPTRITRLQE KEDLQELNDR LAVYIDKVR S ) U 60 51 LELENAGLRL RITESEEVVS REVSGIK AAY EAELADAR KT LDSVAKERAR U 150 HSP90 80 BlHbb (45h) LMNA 101 LQLELSKVRE EHKELKARNA KK EADLLAAQ ARLKDLEALL NSKEAALSTA LMNA RW bl RW 50 WDVhRXW (+1h) (A ry 100 [ 151 LGEKR NLENE VRDLRAQVAK LEGALSEAKK QLQDEMLR RV DAENRLQTLK

bl 201 EELEFQKNIY SEELR ETKRR HETR LVEIDN GR QQEFESK L AEALQDLR RQ ry (A 60 50 ȕDFWLQ ,PPXQR 40 [ 251 HEDQIRHYRD ELEKTYGAKL ENAKQSAERN SSMAGAAHEE LQQTHIRIDS HQVLWRPHW JH2AX D 0 301 LSAELSQLQK QLAAKEAKLR EVEEALSRER EGGRRLLAEK EREMAEMRAR 40 [ 90 kDD 73 kDD 351 MQQQLDEYQE LLDIKLALDM EINAYRKLLE GEEEREERLRLSPLRLSP SPSSQRGARSSPSSQRGARS 30 401 SSGLQHSGAGSGLQHSGAGS AKKRRLEDGEAKKRRLEDGE GREGREGRTSGREGREGRTS FSHHARTSGRFSHHARTSGR VGVEEVDLEGVGVEEVDLEG [ 160 ,PPXQR ii 451 RAGQAVTIWARFFVRLRNKSNVRLRNKSN EDQALGNWQVEDQALGNWQV KRQNGDDPPLKRQNGDDPPL TYRFPPKFTLTYRFPPKFTL KAGQAVTIWA 20 [ 110 20 501 SGAGATHSPPSGAGATHSPP SDVVWKAQSSSDVVWKAQSS WGSGDSLRTAWGSGDSLRTA LINSNGEEVALINSNGEEVA MRMRKLVRTVIIKLVRTVII

HQVLWRPHW 80 JH2AX 551 NNDDDEDEEDDDDDEDEEDD EVSIHHREVSIHHRHHHHHH SGCSGSADPASGCSGSADPA EYNLREYNLR SRSR TVLTVL CGTCGQPADKCGTCGQPADK

15 D 0 60 601 GSAAAASSASGSAAAASSAS SASTVTVSRSASTVTVSRG YRYR SSGGGIGESSGGGIGE GLLGRGLLGRSYVLGSYVLG GAGPRRQAPAGAGPRRQAPA 651 PPQGCSIMQGCSIM +HDG ĮKHOLFDOURG TDLOGRPDLQ 73 42 17 50

40 160 MW rDQJH (kDD) 110 iii 30 80 &RRPDVVLHEULOOLDQWEOXH 20 Housekeeping proteins BlHbb (1h) 15 BlHbb + Tr[L (1h)

XH 15

) 15

60 XH 15 y ) QV U bl QV U bl 50 LDQW LDQW ll y (A 10 QV

ll 10 y (A

L 10 LQWHQVLW

40 L

DMSO br br WR

&RRPDVVLHEULOOLDQWEOXH 30 SHF

LQWHQVLW 05

P 05

LQWHQVLW 05 S r l l

20 R N RWD DVV RWD

15 T RRPDVVLH T M 00 RRPDVVLH 00 00 C C ) ) ) ) ) ) ) ) h h h) h h h h ) ) ) ) ) 5 5 5 5 h 1) 8 5 2 0) (2 +1 (1 (1h) 2 1 (0 (0 (4 ( (+3h (2 ( ( (1 bb (1h)bb bb (3h W b 2 0 (3 Q H H H XW X SO (6 M (7 b [L 7 lLQ bb l bb R R M lH B1 B X lH Bl Bl B lH h h B N N P b V V D + O DMSO (1h) + Tr rot (# pept)M DFWL DMSO B B D D P L LM HS WX E W W bb GAPDH (15 E lH B %HDWLQJLVUDSLGO\SHUWXUEHGE\P\R,,PRGXODWRUVEXWQRWE\ORZGRVHV X0 RI02WUHDWPHQW D i Myosin-II inhibition Rescue BlHbb (25 PM) E ¶0LVP MO MO DLQ 006 MYK (1PM) CWrl LMNA r

f i

VW PM: 10 2 10 30 10 (48h) ii H DMSO CWrl N'D r 006 LMNA 73 MOLMNA 004 JH2AX 17 f MOCWrl H

HDWLQJ QV DLQ 42 r ȕDFWLQ r 004 b AR / AR VW

WDVhRXW +OM

' 002 LVVXH )

T 002 AR / AR * U HDWLQJ WR LMNA '

000

*** B JH2AX +BlHbb

(A * P r 100 rl 15 W 000 ***

ii C QR MO 80 ry CWrl 10 100 h MO PLQ (BPM) 60 WDVhRXW QV

05 ** / 50 PLQ 40 +OM +BlHbb (BPM) HQVLWRPHW

/ *** HDWV PLVPDWF D 00 b 0 5' 0 10 20 30 HDWV 20 0 10 20 30 b MO FRQFHQWrDWLRQ (PM) MO FRQFHQWrDWLRQ (PM) 0 0 1 2 3 4 5 6 7 8 TLPH (hr)

TUDQVFrUHJXODWLRQE\5ARU $*1UHVXOWLQDQWLFRUUHODWHGFKDQJHVLQ/01ADQG'1AGDPDJHDIWHUGD\VRIWUHDWPHQW

FG5HWLQRLFDFLG 5$ LMNA 8QWU RA AGN $QWDJRQLVW $*1 /RZ 0HG +LJK /RZ 0HG +LJK /RZ 0HG +LJK LMNA 73 037LPH K 72 HSP90 90

R2 UQWr 09988 15 5 * ) 20u10 iii RA 09946 r U

8QWU RA AGN 8QWU RA AGN QW 15 10 5 AGN 09909 HSP90) u V 10 LMNA 73 LMNA 73 U WR HSP90 90 10u10 5 P

r 05

4 R 50 10 v (LMNA JH2AX 17 JH2AX 17 u N LMNA (A 42 0 RSH 00 ȕDFWLQ Sl 42 04 05 05 05 05 ȕDFWLQ 0 5u10 1u10 2u10 2u10 3u10 UQWr RA AGN HSP90 (AU) 15 LDPLQA 20 * H

(3h) * DMSO JH2AX QV (72h) QV QV QV 15 20 RA RW RW Day 0 Day 3 (post-treatment) 10 f QV f bl bl H AGN r H HDWHG 15 QV r HDWHG r r 10 XQW

XQW 10

1 DMSO 05 WR

WR P ,PPXQR ,PPXQR 05

r AR / AR 05 +BlHbb AR / AR P ' P r QR r 00 00 00 QR UQWr RA AGN QR UQWr RA AGN 0 1 2 3 4 0 12345 10 70 72 75 TLPH (VHF) TLPH (h) Figure S2 /01$.'RU$)0SURELQJRIQXFOHLFDXVHPLVORFDOL]DWLRQRIPXOWLSOHUHSDLUIDFWRUVZKLFKSHUVLVWVIRUaKRXUV

A iiiEx vivoWLVVXHLQWDFW(KHDUWV VK/01$$OXQJFHOOOLQH B 2YHUOD\ %LQDU\ 'LDVWROH 6\VWROH %OHEE :DVKRXW 0 2 4 24 QV KL36&0V 20 * V ll

60 + ,PSL f

12 H r FH

12 DNA y / KU80 L]DWLRQ l HD Z

40 V RFD /Ar QV ll l 10 HFWHG DNA f 10 FH HD ** LQWHQVLW 10

P r PLV

20 DQV DJPHQWHG Ar R r l W N

/ fr PHDQ XF Z 08 08 0 N 0 0 2 4 6 8 10 0 2 4 24 L r L O b h S S S S b H [ TLPH (VHF) TLPH (h) M lH DV ,P Lf ( D B W + + h + M 80 NLS) U Vh DV h K D W DV W W (QRQ

C D Q1 20 *)3.8 iii8 R2 = 09145 P&KHUU\F*$6 826RVWHR

r = 09563 l VLLMNA QV QV 18 BHfRrH 6 WRS # 10 AfWHr RFL ) /Cy l

U Cytopl. 05 4 16 VLLMNA + OM

XF ***

y (A NT *)3.8

00 H2AX f

γ VLCWl 2 14 BHfRrH VLLMNA + BlHbb KU80 N

LQWHQVLW 10 AfWHr

H v 12 05 0 DWL l NT VLCWl VLLMNA VLLMNA 04 06 08 10 12 H R P&KHUU\F*$6 00 +BlHbb KX80 NXFl/CyWRSl %HIRUH 'XULQJ $IWHU PLQ 0 20 40 DLVWDQFH (μP) F ) U QV E 53BP1 RPA2 DNA 10000 i y (A LQWHQVLW KL36&0V 5000 l KU80 ) 4 RWD U T

0 3 10 NT VLCWl VLLMNA VLLMNA

y (A +BlHbb Cytoplasmic 05 LQWHQVLW

H 00 G R 0 20 40 60 80 μ (WRSRVLGH :DVKRXW DLVWDQFH ( P) KL36&0RUJDQRLG 0 24 ii 7LPH K PLQ PLQ PLQ *)3%3 826RVWHR /HQJWK aPP .\PRJUDSK aVHF Figure S3 .'RI/01$RUUHSDLUIDFWRUVFDXVHVLQFUHDVHG'1$GDPDJHZLWKDFFXPXODWLRQLQODWHFHOOF\FOHUHVFXHE\EOHEELVWDWLQ A i 2N = 92 4N = 8 VLCWl ii iii 50 VLCWl 80 .ROPRJRURY±6PLUQRYWHVWĮ VLKX80

0 # VLCWl VLLMNA VLKU80 VLBRCA1 VLRPA1 VLCRPbR 60

2N = 84 4N = 16 VLLMNA VLCWl RFL VLLMNA **** 40 50 VLKU80 **** **** VLBRCA1 **** **** QV H2AX f VLRPA1 **** **** ** * γ 20 0 VLCRPbR **** QV **** *** ***

2N = 85 4N = 15 VLKU80 0 50 0 5000 10000 15000 20000 KU80 QXFl LQWHQVLWy (AU) DWLRQ l 0

SRSX 2N = 78 4N = 22 VLBRCA1 D

f 50 R

iii80 10000 S WT ) VLLMNA 0 U VLLMNA + BlHbb 1000 60 2N = 81 4N = 19 VLRPA1 DWLRQ y (A

G1 l 50 100 G2 40 SRSX * *** f LQWHQVLW R

0 U 10 20

2N = 59 4N = 41 VLCRPbR (G 50 1 0 0 1 2 3 4 G1 S G2 TRWDl DNA (AU) 0 1 2 4 NRrP WRWDl DNA FRQWHQW (AU)

2 '1$UHSDLUIDFWRUV .85$3 LQWHUDFWZLWK/01$ 2N ** .8SUHIHUHQWLDOO\LQWHUDFWVZLWKQRQSKRVSKRU\ODWHG/01$YVSKRVSKRVROXELOL]HG/01$ 4N )

U E iii

RFL KU70 KU80 LMNA ĮGFP:GFPKU70 1 GFPSG DNA (A

) ,JG

SG WR 10 QV QV H2AX f H2AX 10 QV U γ *** P r LMNA y (A

QR 52 253 LMNB2 10 9 KU80 0 RPA1 7 LQWHQVLW VLCWrl VLLMNA TMPO 10 8 193 … MS MS 2

10 7

Į,JG 88 l l l l l l WD WD D WD 20 WR WR WRWD WRW WRWD WR C DMSO G G G llGRZQ ,J llGRZQ ,J llGRZQ ,J X GFP X GFP X GFP BlHbbLVWDWLQ P P P 15 QV QV RFL 10 H2AX f H2AX γ 5

0 VLCWUO VLCRPbR Figure S4 /01$ EXWQRW/01% LQFUHDVHVZLWK&RO WLVVXHVWLIIQHVVIROORZLQJDµORVHLWRUXVHLW¶PRGHORIWHQVLRQVXSSUHVVHGWXUQRYHU

)ROG FhDQJHUHOWRURZPHDQ AB C ( ( ( 0: N'D 11 10 COL1A2 12581 0.750 (18* Lamin-B1 ~ Const.

y Lamin-A ~ E t y VTN 5165 Lamin-B2 ~ E 0.25 9 FBLN2 13232 10 10 10 COL1A1 1375 LQWHQVLW LQWHQVLW (18* LUM 38642 A B (10 (4 (6 POSTN 96081 9 LFQ LFQ 10 (10 LFQ FBLN1 78137 DPLQ DPLQ (&0

L (6 L FBN2/3 32463 SHF SHF

S FN1 27325 S 8 10 LAMB1 16851 DVV DVV MFAP1 51969 M (4 2 M R = 0.9963 R2 = 0.9687 (LN 63696 10 8 7 10 COL6A3 33959 1 10 1 10 TLVVXH VWLffQHVV EW (kPD) TLVVXH VWLffQHVV EW (kPD) PXN 61 VCL 117 D i 4 ii ) TLN1 272 U

RDX 68 2 ) 3 $GKHVLRQ TLN2 272 F (A U

LMNA , * ZYX 59 RW bl by 4 (A (

DWLR ACTB 41792 WR

1 ACTC1 42019 ,PPXQR P r LMNB r A:B MYH9 2265 1 QR MYH7 22182

DPLQ ABL,M1 83305 L MYH15 2236 0 0 0 2 4 6 8 10 12 14 (4 (6 MYH10 22904 (PbryRQLF DJH (GDyV) MYOM1 18215 MYBPC1 14079 EF ACTN2 10283

) 10 ACTN1 10149

40×10 0.52 U MYO18A 23199 RDW WDLl CRl1D1 ) Et ~ D RDW WDLl CRl1D2 6DUFRPHUH$FWRP\RVLQ PDL,M5 64091 y (A 10 L,MA1 85113 30×10 CRllDJHQ,> (kP W 10 (18* E MYH3 22281 ACTN4 10421 LQWHQVLW 10 20×10 QHVV (10 ff MS MS VWL

LMNA 73 2 10 R = 0.9975 LMNB1 67

VSHF 10×10

LVVXH α LMNB2 68

04 065 /DPLQD

S (6 T fRr R2 > 095 2 (4 DVV R =09995 1 01[ 10[

M 0 6 7 8 9 0 100 200 300 10 10 10 10 CRllDJHQ, CRllDJHQ VSLkHLQ (μJ) MDVV VSHF LFQ LQWHQVLWy

G (WLVVXH %UDLQ +HDUW /LYHU N'D HSP90 84 Lamin-A 73 /DPLQ% 68 6 QV QHVV ff ) 4 D VWL

* (kP W 2 E WLVVXH

10 0 ( BrDLQ HHDrW LLvHr Figure S5 5HGXFWLRQRIVWUHVVLQFUHDVHVSKRVSKRURI/01$DWPXOWLSOHVLWHV 'UXJSHUWXUEDWLRQVGRQRWDIIHFWUHSDLUIDFWRU53$ RU003OHYHOVDFWLYDWLRQ A &RO¶DVH 7U[L 003L &'.L B ) 15 -- ++ ++ + %OHEELVWDWLQ N'D i U QV LMNA 73 10 S6HU 73 y (A

S6HU 73 RPA1 05 HSP90 84 LQWHQVLW MS ) 00 U

) 20 1 QV ii QV

QV U ** * 15 *** y (A

LMNA (A 10 05 MMP2 3 SSHr22 / LMNA R LQWHQVLW

Pr 05

) *** SSHr390 / LMNA DWHG U MS

ryl * DMSO BlHbb CRl'DVH Tr[L MMPL CDKL R * * * h * * * QV 1 + BlHbbLVWDWLQ RVS * LMNA (A LMNA Ph 05 iii + BlHbbLVWDWLQ %OHEELVWDWLQ -- +++++ DMSO BlHbb CRl'DVH 7U[L 003L &'.L &RO¶DVH 7U[L 003L &'.L N'D 3UR003 75 &OHDYHG 68 MMP2 42 C ȕDFWLQ ) %OHEELVWDWLQ 15

U QV (A MMP2

RW 10 R bl Pr

LMNALQWDFW / 05 HG v LPPXQR HD 00 Cl )XUWKHU by DMSO BlHbb CRl'DVH Tr[L MMPL CDKL IUDJP + BlHbbLVWDWLQ SLMNALQWDFW

SLMNAfrDJP )UDFWLRQDODEXQGDQFH

TLPH Figure S6 &0VFXOWXUHGRQVRIWVWLII&RO,OLJDQGJHOVPLPLFEDVLFPRUSKRG\QDPLFVRIin vivoGHYHORSPHQW 0DMRUD[LVDQJOHGLVWU 90 120 60 ( (

ABBlHbbLVWDWLQ C 150 30 035 6 &HOOFHOODOLJQPHQW i *** DMSO CWrl in vitro TUBA1C (459 kDD) TUBB7 (4967 kDD) 180 0 0 BlHbb + GM (4 CMV H l 030 (4 TTLL12 (7281 kDD) BlHbb + ARP ) 210 330 DQJ (COV)

DFWLRQ (6 in vivo ((4 (10) 4 240 300 fr 025 270 TTL D[LV y (A LDWLRQ HD

r r

r 10 8 0.5x 1.7x R D j D

LQV

020 l QV f v PD X WR R *

b LQWHQVLW * 2 (10 X ff /Cy T HXV * l l 015

MS MS 1.7x XF XF FRH N N 001 1 / * 000 0 01 1 10 100 10 6 10 7 5 10 15 0 5 10 15 Rigid (PbryRQLF DJH (GDyV) (PbryRQLF DJH (GDyV) GHl SWLffQHVV EJHl (kPD) plastic

ii 5 D 350 E in vitro (4 CMV

in vivo ((4 (10) ) * 3 300 P ) 4 μ H 6SUHDGLQJ r *** * ( 3RODUL]DWLRQ WX l (ORQJDWLRQ

FX a25 XPH

l 250

3 R ** $[LDODOLJQPHQW r v 0\RILEULODVVHPEO\ ll AR ( HD H l 200 (&0V /DPLQ$ C 2 (10 XF RQJHOV 6RIW 6WLII (6 N 100 LQYLYR ( ( (4 1 0 01 1 10 100 10 6 10 7 0 5 10 15 Rigid (PbryRQLF DJH (GDyV) GHl SWLffQHVV EJHl (kPD) plastic

/01$OHYHOVGRQRWFRUUHODWHZLWKG\QDPLFEHDWLQJLQVWHDGLQFUHDVHVZLWKEDVDOLVRPHWULFWHQVLRQ

F Embryonic heart stiffness G H Rigid plastic i 012 2 i ** * CHll ) 015 * f U NXFlHXV H r QV

DLQ SlRSH a 07

r 1 kPD (A f

VW 008 H

DWLR * 010 1 AR / AR / AR 10 kPD Δ 34 kPD HDWLQJ A:B r A:B

b 004 AR / AR / AR HXV l Δ P 005 r XF

r = 09097 DPLQ R N +BlHbb L

N 2 03 kPD R = 08276 000 0 DMSO OM MYK 000 000 005 010 015 01 1 10 100 CHll ΔAR/ ARrHf ii ii 15 ) f 006 * 34 kPD U H r HD (A 10 10 kPD 1 kPD DWLR / Ar 004

HD 03 kPD Ar A:B r A:B 05 Δ 002 ll H +BlHbb r = 02199 C DPLQ

L R2 = 004837 000 00 01 1 10 100 000 005 010 015 CHll ΔAR / ARrHf iii 25 * 20 V ll FH 15

HDWLQJ 10 b 5 QV +BlHbb

0 01 1 10 100 GHl VWLffQHVV EJHl (kPD) Figure S7 3KRVSKRPLPHWLF6(/01$PXWDQWLVPRUHQXFOHRSODVPLFDQGPRUHUDSLGO\GHJUDGHGWKDQ6$

anti-GFP [L [L B C *)36( i + Tr ( ( *)36( 003L A S22A S22A + TrS22 S22 + DMSO + 7U[L i 15 GFP GFP GFP GFP ) GFPS22A 160

U GFPS22A + Tr[L *)3 GFPS22( LMNA/C 110 80

y (A GFPS22( + Tr[L 36$ 10 LMNA DQWL/DPLQ$& LMNC 60 4000 GF )

* U LQWHQVLW 50 3000 05 y (A

40 A/C GFP 2000 36( P r R DPLQ GF 30 LQWHQVLW N L 00 1000 0 5 10 15 l 20 H

15 R DLVWDQFH (μP) 0 GFPS22( GFPS22( ii + MMP2L ii iii 15 20 *)3/01$& ) 12 β

GFPS22A ry DFWLQ *** GFPS22A U

GFPS22A + Tr[L )

DWLR QV GFPS22A + Tr[L

r 10 GFPS22( U

LMNC 15 GFPS22( y (A GFPS22( + Tr[L

10 (A 08 GFPS22( + Tr[L QV DPLQD

GHQVLWRPHW LMNA /L 10 06 DFWLQ LQWHQVLW

β FrDJPHQWV A/C DVP WR l 05 04

GFP 05 P r DPLQ HRS l P

L 02 r QR XF R N N 00 00 DQWL 00 0 2 4 24 11080 60 50 40 30 20 15 [L ( [L S22A S22 Tr TLPH (h) + MRlHFXlDr ZHLJhW (kDD) ( GFP GFP S22A + Tr S22

GFP GFP

0HWDDQDO\VLVRISXEOLFWLVVXHRPLFVGDWDEDVHVVXJJHVWVSRWHQWLDOµXQLYHUVDOLW\¶RI/01$YV&RO(&0FRUUHODWLRQV

D α 7LVVXHVWLIIQHVV mRNA ~ Col1 mRNA

MRXVH hHDrW GHvHlRSPHQW 10 C R

LL HW Dl Physiol Genomics 2014 CRl XlLQkHrV MMPV CyWRVkHl N( LDPLQD TrDQVFrLSWLRQ rr H l CRl1D2 *** fDFWRrV DWLRQ LR[ 5 *** LPQD v V ) H[SRQHQW

Col1a1

LPQb1LPQb2> VWL V 05 ** ff LQJ QHVV l > v

mRNA ** ( VFD *** * 2 ** ** * 4 * mRNA RJ l DJH α r H

Av 00 QV (75 HPbryR (185 2 x 3 2 1 3 2 2 c l l4 1 p 1 Ttl n1 na rg p1 4 Lo xl1 x x m p a u arb a a o o g Emdm RaraR R ol1a L Loxl2L Lo T MmpMmp8m Mm Mmp9Acta Myh9 S Sun2 L Xrcc6 Y Gata4 lRJ2(Col1a1) C M Tub Lmnb1Lmnb2 Nkx2.5

Chicken E18 embryo RRbHr HW Dl Development 1989 SRlRvHL HW Dl Cell 2013 E F 30 UHbbLQJ HW Dl CRl1D2 E18 Hearts Mol Biol Evol 2015 LPQD 10 9 y) KLGQHy BrHDVW PXVFlH BrDLQ HHDrW GD 25 9 LLvHr 10 y y F2 LXQJ HVVLRQ

a M5 RQLF r F3F5 20 bry

BXrVD fDbrLFLL LQWHQVLW H[S LQWHQVLW THVWLV HHDrW H HP

OvDrLHV bl MS MS 15

A ( CMV 8 LXQJ 10 KLGQHy P r M4 8 R 10 MS LFQ LFQ MS HWHFWD

SSlHHQ N F1 10 M3 DPLQ MHVHQFhyPH BrDLQ 2 D

Lamin-A not M2 R = 08547 f l LLvHr detected in brain R2= 09967 R R2=0.8673 5 10 8 10 9 10 9 5 0 5 10 15 20 CRl1D1 CRl1D1 TLVVXH VWLffQHVV EW (kPD) MS LFQ LQWHQVLWy AGDSWHG frRP SZLfW HW Dl Science 2013 Developmental Cell Previews

Lamin-A Mechano-Protects the Heart

Ioannis Xanthis1 and Thomas Iskratsch1,* 1Division of Bioengineering, School of Engineering and Materials Science, Queen Mary University of London *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.05.041

In this issue of Developmental Cell, Cho et al. (2019) ﬁnd that lamin-A levels in the nuclear envelope are regulated in response to mechanical stimuli to prevent the nucleus from rupture, keep DNA repair factors in the nucleus, and consequentially ‘‘mechano-protect the genome.’’

The cardiac microenvironment constantly ported by LMNA knockdown, which led kinase (CDK) inhibitor and blebbistatin changes during development and dis- to nuclear rupture and increased DNA also resulted in a rescue of LMNA levels, ease, and the associated mechanical damage. To further explore the link be- implying a role for CDKs as regulators of properties affect cellular responses, tween extracellular matrix (ECM) stiffness, LMNA turnover. CDKs were previously such as proliferation or differentiation nuclear integrity, and DNA damage, the shown by the authors to phosphorylate (Ward and Iskratsch, 2019). The stiffness authors first knocked down LMNA in LMNA, which regulated the targeting of of the microenvironment is sensed by embryonic hearts. This led to the mis- LMNA from the nuclear lamina to the the resident cells either through cell-sur- localization of fluorescently tagged DNA nucleoplasm and its subsequent proteo- face receptors and attached adaptor repair factors outside of the nucleus. lytic degradation (Buxboim et al., 2014). proteins, such as talin, or directly by the Further, when human induced pluripotent Consistently, the authors detected an in- nucleus (Pandey et al., 2018; Swift et al., stem cell-derived cardiomyocytes (hIPS- crease in phospho-LMNA fragments after 2013). Previous work from the Discher CM) were cultured on either stiff or soft blebbistatin treatment, but a reduction lab demonstrated that the nuclear enve- matrices, DNA repair factors were again after simultaneous treatment with a pan- lope composition and especially the mis-localized on stiff surfaces due to MMP or a MMP2 inhibitor. lamin-A (LMNA):lamin-B ratio is not only nuclear envelope rupture, which was These experiments linked CDKs to regulated downstream of mechanical sig- aggravated by LMNA knockdown. Im- proteolytic degradation of LMNA by nals, but can also modify differentiation portantly, in both scenarios the effects MMP2 and also to the regulation of nu- pathways (Swift et al., 2013). Moreover, were blocked by simultaneous blebbista- clear stiffness and DNA integrity. Im- recent work from migratory cells high- tin treatment, underlining the role of portantly, immunoprecipitation combined lighted how nuclear lamina composition contractile forces in this process. Last, with MS additionally indicated that repair (and stiffness) can affect nuclear defor- cyclic (passive) stretching of cardiomyo- factors interacted preferentially with mation and lead to DNA damage, thus cytes and U2OS cells also led to DNA non-phosphorylated LMNA compared to linking mechanical signaling to genomic damage and mis-localization of DNA its phospho-solubilized nucleoplasmic instability (Denais et al., 2016; Raab repair factors especially after LMNA form, suggesting that LMNA in its unphos- et al., 2016). However, while previous knockdown, confirming the link between phorylated nuclear lamina-associated studies suggested that DNA damage mechanical forces, LMNA levels, and form also acts as scaffold for the repair can be a factor leading to heart failure nuclear damage. factors. (Higo et al., 2017), how stiffness, nuclear To study how LMNA levels were This involvement of CDKs in stiffness- mechanics, and DNA integrity are all regulated downstream of mechanical dependent phosphorylation of LMNA is associated in the context of heart devel- signaling, a range of mass spectrometry intriguing in several regards. First, CDKs opment or disease remained unresolved. (MS) analyses was performed. The au- were previously shown to affect the In the current issue of Developmental thors first confirmed previous results localization of other nuclear envelope pro- Cell, Cho et al. (2019) use a wide range showing that LMNA levels and collagen-I teins, such as SUN, KASH5, and lamin- of techniques and experimental models are tightly linked to the changing stiffness C2; however, the mechanism remained to analyze the role of LMNA in cardiomyo- of the heart during development. Next, the elusive (Viera et al., 2015). Could it be sec- cyte mechanosensing. authors profiled the proteome of embry- ondary to the loss of LMNA or are these The authors initially set out to study the onic hearts after acute (1 h) drug treat- regulated in a similar manner? Second, interference of cardiomyocyte contrac- ments. Levels of collagen-I, LMNA, and levels of certain endogenous CDK inhibi- tility and collagen matrix rigidity in embry- vinculin were decreased after blebbistatin tors (especially p21 and p27) were previ- onic chick hearts, using pharmacological treatment, but LMNA and collagen-I were ously found to increase during heart inhibitors, collagenase, or transglutami- rescued after simultaneously treating with development, but significantly decreased nase. The authors detected significant a matrix metalloproteinase (MMP) inhibi- in heart disease (Burton et al., 1999). This effects on LMNA levels and concurrent tor, suggesting that MMPs can degrade could suggest that LMNA degradation DNA damage, suggesting a link between both collagen in the ECM and LMNA in due to its phosphorylation by CDKs con- mechanical forces and genome integrity the nucleus. Intriguingly, the simultaneous tributes significantly to disease progres- in the heart. This hypothesis was sup- treatment of cells with a cyclin-dependent sion and heart failure. Finally, the findings

Developmental Cell 49, June 17, 2019 ª 2019 Elsevier Inc. 821 Developmental Cell Previews

Embryonic Heart Healthy Adult Heart Diseased Adult Heart

Cyclin DNA Repair Factors CDKi CDK

Cyclin Cyclin CDK CDK P P P P P P Contractility MMP2 Stiff Nucleus Contractility MMP2 NUCLEAR Contractility Low Actomyosin Soft Nucleus No Rupture High Actomyosin No Rupture Soft Nucleus RUPTURE High Actomyosin

ECM (Col1 etc.) Integrins Talin Vinculin LMNA DNA Repair Factors Sarcomeres

Figure 1. Nuclear Mechanosensing through LMNA and Potential Implications in Heart Disease The stiffness of the ECM changes during development. Simultaneous increases in LMNA in the nuclear lamina, due to changes in expression and turnover, stiffen the nucleus and prevent nuclear rupture. Endogenous CDK inhibitors are strongly reduced in heart disease, leading to higher CDK activity, removal of LMNA from the nuclear lamina, and its degradation in the nucleoplasm by MMP2. The softer nucleus is unable to resist the contractile forces, leading to nuclear rupture and loss of DNA repair factors, which are otherwise associated with LMNA. by Cho et al. could also have implications anism. How CDKs or MMP2 are regulated Higo, T., Naito, A.T., Sumida, T., Shibamoto, M., for cancer therapy and explain the cardio- through cytoskeletal contractility or stiff- Okada, K., Nomura, S., Nakagawa, A., Yamaguchi, T., Sakai, T., Hashimoto, A., et al. (2017). DNA sin- toxicity of doxorubicin and perhaps other ness, however, remains unresolved. The gle-strand break-induced DNA damage response chemotherapeutic agents, since CDK ac- response to blebbistatin is fast, thus causes heart failure. Nat. commun. 8, 15104. tivity in the heart was increased due to excluding transcriptional regulation and Pandey, P., Hawkes, W., Hu, J., Megone, W.V., doxorubicin chemotherapy (Xia et al., rather suggesting a more direct mechani- Gautrot, J., Anilkumar, N., Zhang, M., Hirvonen, 2018). According to Cho et al., this would cal activation. Therefore, evidence points L., Cox, S., Ehler, E., et al. (2018). Cardiomyocytes sense matrix rigidity through a lead to increased phosphorylation and to cardiomyocytes sensing matrix rigidity combination of muscle and non-muscle myosin degradation of LMNA, hence a softer nu- through a network of mechanosensitive contractions. Dev. Cell 44, 326–336.e3. cleus, more nuclear rupture, and DNA proteins, including talin at integrin adhe- Raab, M., Gentili, M., de Belly, H., Thiam, H.R., damage, possibly explaining the link to sions and LMNA in the nuclear envelope. Vargas, P., Jimenez, A.J., Lautenschlaeger, F., heart failure (Xia et al., 2018). Indeed, However, the intermediate molecules Voituriez, R., Lennon-Dume´ nil, A.M., Manel, N., and Piel, M. (2016). ESCRT III repairs nuclear enve- treatment with CDK inhibitors preserved and interactions remain to be elucidated. lope ruptures during cell migration to limit DNA the heart function. damage and cell death. Science 352, 359–362. In summary, Cho et al. provide strong REFERENCES Swift, J., Ivanovska, I.L., Buxboim, A., Harada, T., evidence that LMNA expression and turn- Dingal, P.C., Pinter, J., Pajerowski, J.D., Spinler, over depends on ECM stiffness and is Burton, P.B., Yacoub, M.H., and Barton, P.J. K.R., Shin, J.W., Tewari, M., et al. (2013). Nuclear (1999). Cyclin-dependent kinase inhibitor expres- lamin-A scales with tissue stiffness and enhances regulated by CDKs and MMP2 (Figure 1). sion in human heart failure. A comparison with fetal matrix-directed differentiation. Science 341, If forces are low, LMNA becomes phos- development. Eur. Heart J. 20, 604–611. 1240104. phorylated by CDKs, then moves to the Buxboim, A., Swift, J., Irianto, J., Spinler, K.R., Viera, A., Alsheimer, M., Go´ mez, R., Berenguer, I., nucleoplasm and is degraded by MMP2. Dingal, P.C., Athirasala, A., Kao, Y.R., Cho, S., Ortega, S., Symonds, C.E., Santamarı´a, D., In embryonic hearts, where the ECM is Harada, T., Shin, J.W., and Discher, D.E. (2014). Benavente, R., and Suja, J.A. (2015). CDK2 regu- Matrix elasticity regulates lamin-A,C phosphoryla- lates nuclear envelope protein dynamics and telo- soft, LMNA is present at low levels in tion and turnover with feedback to actomyosin. mere attachment in mouse meiotic prophase. part due to changes in expression Curr. Biol. 24, 1909–1917. J. Cell Sci. 128, 88–99. (through self-regulation via the retinoic Cho, S., Vashisth, M., Abbas, A., Majkut, S., Vogel, Ward, M., and Iskratsch, T. (2019). Mix and (mis-) acid pathway [Swift et al., 2013]), but K., Xia, Y., Ivanovska, I.L., Irianto, J., Tewari, M., match - The mechanosensing machinery in presumably also in part because of CDK Zhu, K., et al. (2019). Mechanosensing by the the changing environment of the developing, lamina protects against nuclear rupture, DNA healthy adult and diseased heart. Biochim. activity, consecutive removal from the nu- damage, and cell-cycle arrest. Dev. Cell 49, this Biophys. Acta. Mol. Cell Res. Published online clear envelope, and subsequent degrada- issue, 920–935. February 8, 2019. https://doi.org/10.1016/j. tion. On the contrary, in adult hearts, bbamcr.2019.01.017. Denais, C.M., Gilbert, R.M., Isermann, P., where the matrix is stiffer, LMNA is stabi- McGregor, A.L., te Lindert, M., Weigelin, B., Xia, P., Liu, Y., Chen, J., Coates, S., Liu, D.X., and lized in the membrane in order to prevent Davidson, P.M., Friedl, P., Wolf, K., and Cheng, Z. (2018). Inhibition of cyclin-dependent ki- Lammerding, J. (2016). Nuclear envelope rupture nase 2 protects against doxorubicin-induced nuclear rupture and DNA damage, in what and repair during cancer cell migration. Science cardiomyocyte apoptosis and cardiomyopathy. the authors call a ‘‘lose it or use it’’ mech- 352, 353–358. J. Biol. Chem. 293, 19672–19685.

822 Developmental Cell 49, June 17, 2019