Mechanosensing by the Lamina Protects against …discher/documents/_______Me..., Kuangzheng Zhu 1,...

Article

Mechanosensing by the Lamina Protects againstNuclear Rupture, DNA Damage, and Cell-CycleArrest

Graphical Abstract

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

Authors

Sangkyun Cho, Manasvita Vashisth,

Amal Abbas, ..., Roger A. Greenberg,

Benjamin L. Prosser, Dennis E. Discher

[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.

Cho et al., 2019, Developmental Cell 49, 920–935June 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, USA4 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 LaminaProtects against Nuclear Rupture,DNA Damage, and Cell-Cycle ArrestSangkyun Cho,1Manasvita Vashisth,1 Amal Abbas,1 StephanieMajkut,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, USA2Orthopaedic Surgery and Cell & Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia,

PA 19104, USA3Center for Systems and Computational Biology, Wistar Institute, Philadelphia, PA 19104, USA4Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA5Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA6Penn Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA7Lead Contact

*Correspondence: [email protected]://doi.org/10.1016/j.devcel.2019.04.020

SUMMARY

Whether cell forces or extracellular matrix (ECM) canimpact genome integrity is largely unclear. Here,acute perturbations (�1 h) to actomyosin stress orECM elasticity cause rapid and reversible changesin lamin-A, DNA damage, and cell cycle. The findingsare especially relevant to organs such as the heartbecause DNA damage permanently arrests cardio-myocyte proliferation shortly after birth and therebyeliminates regeneration after injury including heartattack. Embryonic hearts, cardiac-differentiated iPScells (induced pluripotent stem cells), and variousnonmuscle cell types all show that actomyosin-driven nuclear rupture causes cytoplasmicmis-local-ization of DNA repair factors and excess DNA dam-age. Binucleation and micronuclei increase astelomeres shorten, which all favor cell-cycle arrest.Deficiencies in lamin-A and repair factors exacerbatethese effects, but lamin-A-associated defects arerescued by repair factor overexpression and alsoby contractility modulators in clinical trials. Contrac-tile cells on stiff ECM normally exhibit low phosphor-ylation and slow degradation of lamin-A by matrix-metalloprotease-2 (MMP2), and inhibition of thislamin-A turnover and also actomyosin contractilityare seen to minimize DNA damage. Lamin-A is thusstress stabilized to mechano-protect the genome.

INTRODUCTION

Proliferation of many cell types slows dramatically shortly after

birth and is absent in key adult tissues (Li et al., 1996), which pre-

sents a major challenge to regeneration. Cell growth in culture is

modulated by the stiffness of the extracellular matrix (ECM),

which generally promotes actomyosin stress (Engler et al.,

2006; Paszek et al., 2005) and affects the conformation (Sawada

et al., 2006), post-translational modification (PTM) (Guilluy et al.,

2014), localization (Dupont et al., 2011), and degradation (Dingal

and Discher, 2014) of mechanosensitive proteins. Actomyosin

links to ECM and to nuclei, such that rapid changes to the

ECM and tissue mechanics that result from chronic or acute

injury or even drug therapies (e.g., cardiac arrest or cardiomyop-

athy treatment [Green et al., 2016]) can in principle affect the nu-

cleus (Takaki et al., 2017; Wiggan et al., 2017; Kanellos et al.,

2015) and perhaps the DNA within. DNA damage and telomere

shortening are indeed well documented in injuries that affect

the heart (Chang et al., 2018; Higo et al., 2017; Sharifi-Sanjani

et al., 2017; Oh et al., 2003), as well as nonmuscle tissue—but

DNA damage and repair are rarely studied in developing organs,

and relationships to proliferation, ECM stiffness, and actomyosin

stress are understudied.

DNA damage and senescence increase with many disease-

linked mutations, including those in the nucleoskeletal protein

lamin-A (LMNA) that forms a structural meshwork around chro-

matin (Turgay et al., 2017; Shimi et al., 2015; Gruenbaum et al.,

2005). LMNA deficiencies associate with elevated DNA damage

(Graziano et al., 2018; Liu et al., 2005) and result in accelerated

aging of stiff tissues similar to deficiencies in DNA repair factors

(e.g., KU80) (Li et al., 2007). Moreover, progeroid syndromes are

caused only by mutations in LMNA and multiple DNA repair fac-

tors, but LMNA’s primary function in development remains hotly

debated (Burke and Stewart, 2013), with supposed roles in gene

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

of cells with LMNA defects is rescued by culturing cells on

almost any ECM (versus rigid plastic [de La Rosa et al., 2013;

Hernandez et al., 2010]) and by treatment with at least one

drug affecting both cytoskeleton and nucleo-cytoplasmic traf-

ficking (Larrieu et al., 2018, 2014). Relationships between lamins,

920 Developmental Cell 49, 920–935, June 17, 2019 ª 2019 Elsevier Inc.

0 1 2 310

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LMNA & DNA damage are mechano-sensitive to myosin-II & matrix, but DNA damage levels change more rapidly

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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

nonetheless obscure—especially in tissues.

Embryonic hearts beat spontaneously for days after isolation

from early chick embryos, and beating is acutely sensitive to

myosin-II inhibition (Figure 1A), as well as enzymatic stiffening

or softening of the ECM (Majkut et al., 2013). The latter studies

reveal an optimal stiffness for beating that is likewise evident

for cardiomyocytes (CMs) cultured on gels (Majkut et al., 2013;

Engler et al., 2008; Jacot et al., 2008). DNA damage is conceiv-

ably optimized in heart as it triggers a switch from proliferation to

senescence in post-natal hearts (Puente et al., 2014). DNA dam-

age is also implicated in telomere attrition and binucleation of

CMs that signal irreversible exit from cell cycle (Aix et al.,

2016). We postulated embryonic hearts with rapidly tunable

mechanics could prove useful as a tissue model for clarifying

protein-level mechanosensing mechanisms in vivo that could

be studied thoroughly in vitro with many cell types.

LMNA is reportedly ‘‘undetectable’’ in early embryos (Stewart

and Burke, 1987), but it accumulates clearly and progressively in

tissues such as heart, bone, and lung (Solovei et al., 2013; Rober

et al., 1989; Lehner et al., 1987) and is highest in adults within

these mechanically stressed, stiff tissues that are collagen rich

(Swift et al., 2013). LMNA deficiencies accordingly produce

measurable defects weeks after birth in such tissues, including

heart (Kubben et al., 2011;Worman and Bonne, 2007). We there-

fore hypothesized that LMNA in normal embryos mechano-

senses the earliest microenvironment in the first organ and

increases not only to stiffen the nucleus (Osmanagic-Myers

et al., 2015; Dahl et al., 2008; Pajerowski et al., 2007) but also

to regulate repair factors that confer resistance to both DNA

damage and cell-cycle arrest.

RESULTS

Contractility and Collagen Perturbations Rapidly ImpactLMNA and DNA Damage in HeartsTo first assess the consequences of cytoskeletal stress on nuclei

in a tissue, we arrested embryonic hearts (embryonic day [E] 4)

with reversible inhibitors of myosin-II (Figure 1A). Spontaneous

beating of hearts stopped in minutes with blebbistatin (blebb),

but the heart re-started just as quickly upon washout of

drug (Figure 1A, right). An activator of cardiacmyosin-II omecam-

tiv mecarbil (‘‘OM’’)—in clinical trials for heart failure (National In-

stitutes of Health, 2016b [NCT02929329])—re-started the hearts

reliably. Quantitative immunoblots and mass spectrometry (MS)

measurements (Figures 1B, 1C-i, S1A, and S1B) revealed a rapid

decrease in LMNA (�45 min decay constant), while LMNB re-

mained unchanged (Figure 1C-i, upper right inset; Figure 1C),

and similar results were evident for a cardiac myosin-II-specific

inhibitor mavacamten analog (MYK)—in clinical trials for hyper-

trophic cardiomyopathy (National Institutes of Health, 2016a

[NCT02842242]). Despite rapid recovery of beating after drug

washout (Figure 1D), LMNA recovered more slowly (>3 h), indi-

cating slow synthesis.

DNA damage in post-natal development with LMNA defi-

ciencies (Liu et al., 2005) led us to hypothesize acute, embryonic

changes in phospho-histone gH2AX, as a primarymarker of DNA

damage. A rapid decrease in DNA damage was surprising with

myosin-II inhibition (Figure 1C-ii) given the decrease LMNA, but

an electrophoretic comet assay confirmed the gH2AX results

(Figure 1D). It is useful to keep inmind that the heart beats reason-

ably well with LMNA deficiencies and mutations. Because blebb

washout recovers beating while LMNA remains low, we antici-

pated a large spike inDNAdamage shortly afterwashout (Figures

1C-i and 1C-ii, right inset). LMNA and DNA damage eventually

reached control levels (in hours), but the spike highlights the

disruptive effects of actomyosin stress on genome integrity.

Actomyosin contractility is generally downstream of ECM

stiffness (Ulrich et al., 2009; Engler et al., 2006), including for

immature CMs (Engler et al., 2008; Jacot et al., 2008). Acute per-

turbations of collagen matrix might therefore be expected to

affect DNA damage. Collagenase treatment for 45-min indeed

resulted in rapid decreases in DNA damage and LMNA (Fig-

ure 1E), consistent with rapid softening of E4 hearts (�50%)

and weaker beating (Majkut et al., 2013). Treatment with tissue

transglutaminase (TGM), a cross-linker of ECM that stiffens

heart and thereby increases basal tension (>2-fold in 2 h [Majkut

et al., 2013]), increased gH2AX and LMNA (only after 3 h) except

when collagenase was subsequently added (Figure 1E). LMNA

thus decreases quickly or increases slowly in response to

changes in ECM stiffness or actomyosin tension, both of which

appear to also affect DNA damage. Effects are also generally

reversible.

DNA Damage in LMNA-Deficient Hearts Perturbs CellCycle and Causes Aberrant BeatingExcess DNA damage has been shown to impact the cell cycle

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 versusMOCtrl, 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 versusG2; or 2N versus 4 N). (ii) Blebbwashout increasesG2 and 4N 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

to assess the biological consequences of DNA damage in

LMNA-suppressed embryonic hearts. Morpholino (MO) medi-

ated knockdown (KD) of LMNA (MOLMNA; �40% KD in 24 h)

was achieved with no significant effect on contractile beating

(Figures 1F-i and S1E). LMNA is thus not primarily upstream of

beating, consistent with knockout mice (Singh et al., 2013).

Although past studies also suggest LMNA is not detectable in

early embryonic hearts and is therefore dispensable (Solovei

et al., 2013; Stewart and Burke, 1987), morpholino KD increased

gH2AX in E4 hearts (Figure 1F-ii). Similar effects were seen by

modest repression with retinoic acid (RA) of LMNA (20% only af-

ter 3 days) that acts transcriptionally (Swift et al., 2013), while RA-

antagonist (AGN) upregulated LMNA and suppressed DNA dam-

age (Figures 1F and 1G). blebb was sufficient to rescue the DNA

damage in MOLMNA KD hearts, supporting our hypothesis that

high contractility coupled to low LMNA causes excess damage.

Bona fide DNA damage can perturb cell cycle (Puente et al.,

2014); the cell cycle inhibitor p21 indeed trended with gH2AX in

MO-treated hearts (Figure 1F-ii). EdU (5-ethynyl-20-deoxyuridine)integration before and after blebbistatin addition and washout

(per Figures 1A–1C) further revealed an increase in cells in G2,

with fewer cells inS, andan increase in 4Ncells (Figure 1G).Given

that acute DNA damage can inhibit cell cycle in developing heart

and is somehowcoupled tomatrix-myosin-lamins (Figure 1H),we

assessed DNA damage that was directly and abruptly increased

with etoposide (4-fold, 1 h) (Figure 2A). This inhibitor of replication

and transcription quickly caused aberrant beating that could be

reversed upondrugwashout (Figure 2B);manycardiac laminopa-

thies exhibit arrhythmias and broader conduction defects (Fatkin

et al., 1999). No such effects were seen with the same dose of

H2O2 (for oxidative stress) relative to DMSO control hearts unless

H2O2 exposure was sustained for days. DNA damage can have

multiple causes, ranging fromdrugs andoxidative stress to nucle-

ases, but reversal of damage fromnatural sources or etoposide in

hours (Figures 1F, 1G, and 2A) suggest an important role for DNA

repair factors (Figure 2C).

LMNA KD in Intact Hearts and Human iPS-CMs: NuclearRupture and Loss of DNA RepairTo address how increased mechanical stress causes DNA dam-

age, the integrity of the nucleus was scrutinized in intact E5

hearts doubly transfected with DNA repair protein GFP-KU80

(XRCC5) and with a cytoplasmic protein that can bind nuclear

DNA, mCherry-cGAS (cyclic GMP-AMP synthase [Raab et al.,

2016]) (Figure 3A-i). Cytoplasmic mis-localization of KU80 and

concomitant formation of cGAS puncta at the nuclear periphery

provided evidence of nuclear rupture in transfected hearts (Fig-

ure 3A-i, yellow arrow), and the fraction of such double-positive

A B

C

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.


cells increased with MOLMNA KD unless blebb was added (Fig-

ure 3A-ii). Myosin-II inhibition disrupted sarcomeres, which

rapidly recovered upon drug washout (Figure 3A-iii), consistent

with beating (Figures 1A–1C); and mis-localization of KU80 and

cGAS showed the same trends (Figure 3A-iv), without any

apparent changes in cell viability (Figure 2A-i). DNA damage

again spiked upon blebb washout and remained excessively

high with addition of a nuclear import inhibitor (Imp-i; ivermectin)

that keeps KU80 mis-localized to cytoplasm (Figures 3B-i, 3B-ii,

and S2A-ii). LMNA immunofluorescence (IF) for MOLMNA and

A D

E

B

C

F

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 uponwashout (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)


blebb/washout hearts (Figures 3B-iii and 3C) confirmed immu-

noblot trends (Figures 1C-i and 1F) and showed Imp-i kept LMNA levels low upon washout.

Human induced-pluripotent-stem-cell-derived CMs (hiPS-

CMs) cultured on soft or stiff gels with collagen-ligand constitute another model for clarifying mechanisms at the single-cell level. Nuclei in hiPS-CMs ‘‘beat’’ (Figure 2B) and express low LMNA relative to some well-studied human cell lines (e.g., lung-derived A549 cells) and relative to mature heart, which is much stiffer than embryos (Swift et al., 2013). Cell morphologies and sarcomere striations resemble those of embryonic or fetal CMs. On very stiff gels (40 kPa) but not on gels as soft as embryos (0.3 kPa), hiPS-CMs exhibited cytoplasmic, mis-localized KU80 (Figure 3D), consistent with envelope rupture under high nuclear stress (Figure 1H). Nuclear blebs typical of ruptured nuclei formed at points of high curvature in such cells (Xia et al., 2018), appearing rich in LMNA but depleted of LMNBs (Figure 3D). Stress-induced nuclear rupture was also imaged as rapid and stable accumu-

lation of mCherry-cGAS upon nuclear probing with a high cur-vature atomic force microscopy (AFM) tip (<1 mm), using nano-Newton forces similar to those exerted by a cell’s actomyosin (Figure 2C).KD of LMNA (siLMNA) doubled the fraction of cells with cyto-

plasmic KU80 (Figures 3E-i and S2D-i) and increased gH2AX foci (Figures 3E-ii and S2D-ii). These observations seem consistent with iPS-CM results for LMNA defects, which show nuclear bleb-bing (Lee et al., 2017), focal loss of nuclear membrane (Siu et al., 2012), cytoskeletal defects (Bollen et al., 2017), and fibrosis, all of which increase with electrical stimulation of contraction. We found LMNA levels in hiPS-CMs exhibited sensitivity to substrate stiffness similar to that in embryonic hearts (Figure 3E-iii), and we saw blebb-treated siLMNA cells on rigid plastic rescued nuclear rupture (Figure 3E-i), as well as DNA damage (Figure 3E-ii). Relaxation of rigidity-driven actomyosin stress can thus limit nu-clear rupture.Cytoplasmic mis-localization of KU80 is merely representa-

tive: repair factors 53BP1 and RPA2 also simultaneously mis-

localized to the cytoplasm upon LMNA KD (Figure 2E-i). Time-

lapse images of GFP-53BP1-expressing cells further revealed mis-localization persists for hours (Figure S2E-ii). However, total KU80 levels did not vary (Figure 2F), suggesting that nuclear retention is key.To assess whether mis-localization of repair factors is a gen-

eral consequence of increased mechanical stress, LMNA KD of both CM and U2OS osteosarcoma cells was followed by cyclic stretch at a frequency and magnitude similar to that of the intact heart (8% uniaxial strain at 1-Hz frequency) (Fig-ure 3F-i). Mis-localized GFP-KU80 increased >2-fold with imposed stretching of MOLMNA KD E4 CMs but not MOCtrl (Fig-

ure 3F-ii), and the fold-increase was even more dramatic with the nonbeating U2OS cells because baseline mis-localization was very low. DNA damage also increased with stretching of MOLMNA CMs while LMNA levels showed an insignificant in-crease (Figure 3F-iii). An acute increase in stress without a pro-portionate increase in LMNA can thus cause mis-localization of repair factors and DNA damage, which could affect tissue-level function (Figures 2A and 2B). Etoposide-treated hiPS-CM orga-noids indeed show aberrant beating (Figure 2G).

Loss of Repair Factors or LMNA: DNA Damage,Binucleation, Micronuclei, and Cell-Cycle ArrestTo assess the impact of a limited capacity for DNA repair

in cells, key individual DNA repair factors were partially

depleted individually and in combination (si-Combo). BRCA1

is implicated in myocardial infarction and ischemia (Shukla

et al., 2011), and RPA1 appeared abundant and constant

in level in our MS of drug- or enzyme-perturbed hearts (Figures

1C and 1E). DNA damage increased in hiPS-CMs for all

repair factor KDs, and the combination proved additive (Fig-

ure 4A-i).

Excess DNA damage in post-natal hearts has been associated

with CM binucleation (Aix et al., 2016)—a hallmark of irreversible

cell cycle exit. Soft ECM suppressed binucleation of embryonic

CMs relative to stiff ECM (Figure 4A-ii) while suppressing mis-

localization of repair factors (Figure 3E-i). Partial KD of DNA

repair factors directly increased the fraction of binucleated

hiPS-CMs, as well as those with micronuclei (Figure 4A-iii),

both of which correlated strongly with gH2AX. Repair factor

KD further increased the fraction of ‘‘4N’’ cells (versus ‘‘2 N’’; Fig-

ures 4A-iv and S3A), in agreement with trends for blebb-treated

hearts (Figure 1G) andwithmitotic arrest. LMNAKD had all of the

same effects, consistent with a role in repair factor retention in

nuclei, and, consistent also with a late checkpoint, KD showed

more DNA damage per DNA in 4 N cells relative to control cells

(Figure 3B). Importantly, with siLMNA or siCombo KD, increased

gH2AX and cell cycle perturbations were evident not only at

steady state but also within 1 h of damaging DNA with etoposide

(per Figure 2)—which partially recovered after drug washout

(Figure 4B).

Myosin-II inhibition once again rescued the DNA damage, as

well as cell cycle effects, in siLMNA KD cells (Figure 4C-i),

whereas siCombo cells maintained high DNA damage (Fig-

ure 3C). Furthermore, overexpression of relevant DNA repair

factors (with GFP-KU70, KU80, BRCA1 = ‘‘GFP-Combo’’)

rescued only the excess DNA damage in nuclear-ruptured cells

compared to non-ruptured (Figure 4C-ii). A baseline level of

damage in the latter cells was not affected, likely because repair

factors become limiting only upon nuclear rupture or depletion. A

combination of factors (GFP-Combo) rescued the excess dam-

age, while one repair factor had no effect on its own (53BP1)

even on the ruptured nuclei. Rescue of DNA damage again sup-

pressed the fraction of 4 N cells (Figure 4B lower) and cells in G2

(Figure 3D).

Since cardiac telomere attrition has been implicated in CM bi-

nucleation (Aix et al., 2016), as well as in cardiomyopathies and

heart failure (Sharifi-Sanjani et al., 2017), we assessed rupture-

associated telomere shortening as a more localized form of DNA

damage (Figure 4D). U2OS osteosarcoma cells with ruptured

nuclei (cytoplasmic anti-KU80) showed lower total intensity of

telomeric repeat-binding factor-1 (TRF1, as an mCherry fusion;

Figure 4E-i) and smaller TRF1 foci size (Figure 4E-ii), as well as

higher gH2AX in these LMNA KD cells. Importantly, partial KD of

either KU80 or multiple repair factors also caused telomere

attrition in cells with normal LMNA levels (Figure 4F). Proper

retention of DNA repair factors within stressed nuclei thus re-

quires sufficient LMNA, but LMNA also interacts with key repair

factors (including KU70/80 and RPA1) as confirmed by immuno-

precipitation-MS (IP-MS) (Figure S3E). IP-MS further indicates


A B

C

D E

G H I

F

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)


A B C

D E

F

GH

I

(legend on next page)


KU80 interacts preferentially with non-phosphorylated LMNA (GFP-wild type [WT]) compared to its phospho-solubilized nucleoplasmic form (per IP by anti-pSer22 or phosphomimetic mutant GFP-S22E) (Figure 4G), suggesting that LMNA scaf-folds the repair of DNA at the periphery—which often includes telomeres (Figure 4H). Importantly, analysis of telomeres in embryonic CMs by quantitative FISH (Q-FISH) revealed LMNA KD causes telomere shortening unless actomyosin is inhibited (Figure 4I). The findings motivate deeper mechanistic insights into how LMNA adjusts to actomyosin stress (Figures 1A–1E, 3C, and 3E-iii) to thereby regulate DNA damage and the cell cycle.

LMNA Effectively Stiffens Nuclei during Development and Increases with Collagen-I ECMTo begin to assess in normal beating hearts how LMNA senses stress, nuclear deformations were imaged at E4—just 1 to 2 days after the first heartbeat (Figure 5A). Transfec-tion of a minor fraction of CMs in hearts with mCherry-histone-

H2B or GFP-LMNA facilitated imaging, and each �1-Hz contraction of heart tissue was seen to strain individual CM nuclei by up to �5%–8%. Recall that 8% stretch at 1-Hz was sufficient to rupture CM nuclei (Figure 3F). While transfec-tion did not affect overall tissue-level beating, GFP-LMNA nuclei deformed much less than neighboring control nuclei (Figure 5A-ii), consistent with in vitro studies showing LMNA stiffens the nucleus (Pajerowski et al., 2007; Lammerding et al., 2006). MS of lysates quantified 29 unique LMNA pep-tides at E4 (Figure 1C), confirming our early detection of func-tional LMNA (Figure 1B) despite past reports (Solovei et al., 2013; Stewart and Burke, 1987), showed a rapid increase in LMNA expression by �3-fold from soft E4 hearts to stiffer E10 hearts (Figures 5B and 5C). B-type lamins remained rela-tively constant, in agreement with studies of diverse soft and stiff adult tissues that suggest LMNA-specific mechanosensi-

tivity (Figures 4A–4C) (Swift et al., 2013). Trends are confirmed by confocal IF and immunoblots of embryonic heart (Figures 5C-i and 5C-ii, insets; Figure S4D).

Collagen-I (a-1 and a2) exhibited the largest fold change in

MS analyses of heart development (Figure 5B), with calibrated

spike-ins of purified collagen-I matching past measurements of

collagen in developing chick hearts (Woessner et al., 1967)

(Figure 4E). Collagen-I becomes the most abundant protein in

developed animals and is a major determinant of tissue stiff-

ness (Shoulders and Raines, 2009); therefore, its increase

together with many other structural proteins (Figure 4C) is

consistent with heart stiffening (Figure 2F) (Majkut et al.,

2013). Mechanosensitive proteins at the interface between ad-

hesions and actomyosin cytoskeleton were also upregulated,

including paxillin (Zaidel-Bar et al., 2007) and vinculin (Huang

et al., 2017), consistent with increased adhesion and contractile

stress (Geiger et al., 2009). Downregulation of protein synthesis

and catenins suggest a transition from a rapidly growing,

epithelial-like embryo to a more specialized, terminally differen-

tiated state.

Lamin-A:B ratio increased with collagen-I (Figures 5C-i and

S4F) and with tissue stiffness Et (Figure 5-ii), with a power law

exponent a = 0.3 (versus collagen-I) similar to that for diverse tis-

sue proteomes (Cho et al., 2017). Experiments fit a ‘‘use it or lose

it’’ mathematical model (Figure 5C-i) wherein mechanical tension

on filamentous collagen-I and LMNA (Dingal and Discher, 2014)

suppresses protease-driven degradation (Figure 5C-i). The plot

versus Et yielded a�0.7 that is similar for adult tissues (a�0.6

[Swift et al., 2013]), with soft E10 brain and stiff E10 liver fitting

well (Figures 4F and 4G). Importantly, 45 min collagenase soft-

ened E4 hearts by �50% and decreased LMNA (Figure 5C),

per Figures 1A–1E.

Different combinations of drugs were used to identify mecha-

nisms for how LMNA levels change in tight coordination with

collagen-I and tissue stiffness. MS-derived heatmaps of drug-

treated E4 hearts (e.g., collagenase and blebb) reveal rapid

and large decreases in collagen-I, vinculin, and LMNA (Figure 5D;

confirmed by immunoblot, Figure 5A). Little to no change was

observed across most of the detected proteome including

DNA repair factors (KU70 and RPA1 [Figure 5B-i]), matrix-metal-

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 mMdoses 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 LMNAwith blebb is rescued byMMP-i’s, includingMMP2-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).


A B

E

C

D

F

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)


5B-iii), cardiac myosin-II (MYH7B), vimentin (VIM), and B-type

lamins. Similar results were obtained even in the presence

of the translation inhibitor (Trx-i) cycloheximide, except for de-

creases in a few cytoskeletal proteins that indicate rapid transla-

tion (e.g., actin [Katz et al., 2012]). Decreased translation thus

does not explain the heart’s decreases in collagen-I and LMNA

with blebb and/or collagenase.

To clarify howmyosin-II inhibition (intracellular) leads to a rapid

decrease in collagen-I (ECM) in intact hearts, a pan-inhibitor of

matrix-metalloproteinases (MMP-i) was used with blebb. The

combination surprisingly prevented collagen-I degradation. In

contrast, a CDK inhibitor (CDK-i; likely affecting CDK4/6) that

limits interphase phosphorylation of LMNA (e.g., pSer22 nor-

malized to total LMNA: pSer22/LMNA in Figure 5A) rescued

blebb-induced decreases in LMNA levels without affecting

the collagen-I decrease. Vinculin’s decrease concurs with

decreased actomyosin tension regardless of MMP-i (Figure 5D),

but MMP-i surprisingly rescued blebb-induced decreases in

LMNA similarly to CDK-i (Figures 5D and S5A).

Mechano-protection of the Genome Is Limited by LMNAPhospho-solubilizationWe hypothesized that one or more MMPs directly degrade

LMNA. Nuclear MMP2 is well documented for many cell types

including CMs (Xie et al., 2017), and LMNA (but not B-type lam-

ins) has been speculated to be a proteolytic target (Baghirova

et al., 2016). Beating E4 hearts treated with MMP-i or an

MMP2-specific inhibitor, MMP2-i, rescued the blebb-induced

decrease in LMNA in immunoblots that were also probed with

a previously untested anti-pSer390 (Figures 5E-i and 5E-ii). For

other phosphosites, interphase phosphorylation, solubilization

(Kochin et al., 2014), and subsequent degradation of LMNA

(Naeem et al., 2015; Bertacchini et al., 2013) are already known

to be suppressed by actomyosin tension on nuclei (Buxboim

et al., 2014; Swift et al., 2013). Intact phospho-LMNA (73 kDa)

increased upon blebb treatment (Figure 5E-iii; pSer390intact/

LMNAintact), but even more dramatic was the suppression by

MMP2-i and MMP-i (regardless of blebb) of a 42 kDa pSer390

fragment (pSer390fragm./pSer390intact) (Figure 5E-iv). Fragment

size matches predictions for MMP2 digestion (Song et al.,

2012) but is likely a transient intermediate that is further

degraded (Buxboim et al., 2014) (Figure 5E-v). MS of hearts

treated with blebb or collagenase not only confirmed the trend

for the fragment (>6 LMNA peptides) (Figure 5F) but also showed

MMP2 levels remain constant, as supported by immunoblots for

MMP2’s activation (cleaved/pro-MMP2 ratio; Figures 5G and

S5B). Anti-MMP2 (for both pro- and active forms) was mostly

nucleoplasmic in isolated CMs with localization unaffected by

actomyosin inhibition (Figure 5H). Nucleoplasmic MMP2 in

neonatal CMs (Baghirova et al., 2016; Kwan et al., 2004) sug-gests that, independent of maturation stage, active MMP2 is localized appropriately for degradation of phospho-LMNA downstream of actomyosin tension (Figure 5E-v).DNA damage was assessed after acute 2-h treatments of

CDK-i or MMP-i plus blebb. Whereas MMP-i had no effect on gH2AX relative to blebb alone, CDK-i plus blebb greatly sup-pressed DNA damage (Figure 5I). Since CDK-i maintains LMNA at the lamina (unlike MMP-i) and thereby stiffens and sta-bilizes the nucleus (per Figure 5A and single-cell measurements [Buxboim et al., 2014]), the results suggest that even low levels of nuclear stress are resisted to minimize both rupture and DNA damage in the embryonic heart.

Isolated CMs Elucidate Effects of ECM Elasticity and Contractility on LMNA Turnover by MMP2Intact heart presents many complications to understanding LMNA regulation and its function. As the heart stiffens in devel-opment, for example, collagen ligand also increases greatly for adhesion (Figures 5B and 5C), sarcomere assembly becomes very dense (Figures 6A and S6A-i), cells and nuclei elongate (Fig-ures 6A-ii and S6B) with stabilization by microtubules (Robison et al., 2016), cells align (Figure 6C), and nuclear volumes decrease (Figure 6D). Gels with constant collagen-I ligand and of controlled stiffness (0.3–40 kPa) (Figure 6B) showed—after just 24 h—the same stiffness-correlated morphology and cyto-skeleton trends for isolated E4 CMs as the intact heart from E4 to �E11 (Figure 6C). Although matrix elasticity had large effects on size, shape, sarcomere assembly, and contractility of early CMs (Figures 6A-i, 6A-ii, and S6E) similar to later stage CMs (Ribeiro et al., 2015; Engler et al., 2008; Jacot et al., 2008), blebb quickly disrupted cell spreading, striation, and elongation on stiff gels (10 kPa) and on collagen-coated rigid plastic (Figure 6C). Gels that mimic the stiffness of E4 heart (1–2 kPa) were optimal for cell beating (Figure 6F), consistent with past studies (Majkut et al., 2013; Engler et al., 2008), and nuclear ‘‘beating strain’’ in isolated CMs (Figures 6F-i and S6G-i) was similar in magnitude to that in intact heart (5%–8%) (Figure 5A). Surprisingly, lamin-

A:B intensity increased monotonically with gel stiffness rather than exhibiting an optimum (Figures 6D-i and S6G-ii). LMNA’s in-crease depended on myosin-II, as did cell/nuclear aspect ratio (AR), spreading, and sarcomere assembly (Figures 6C, 6D-ii, and S6A,E). Monotonic increases with stiffness of ECM agree with elevated isometric tension as documented for other cell types (e.g., Engler et al., 2006). Remarkably, MMP-i or MMP2-i rescued the blebb-induced decrease in lamin-A:B at the sin-gle-cell level (Figure 6D-i) as seen for intact embryonic hearts (Figure 5D-i) despite no significant effects on cell morphology or 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 raremitotic 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) % 4N cells are highest in GFP-S22E cells, but blebbminimizes 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).


Since myosin-II-dependent increases in lamin-A:B versus gel

stiffness for isolated CMs (Figure 6D-i) align with lamin-A:B’s in-

crease with heart stiffness and contractility during development

(Figures 5B and 5C), LMNA phosphorylation was re-examined

(per Figures 5D and S5A). IF with anti-pSer22 and pSer390

proved consistent with tension-suppressed LMNA phosphoryla-

tion and turnover: cells on soft gels showed high nucleoplasmic

phospho-signal as did cells on stiff gels treated with blebb

(Figure 6E). The latter also showed cytoplasmic phospho-

signal, consistent with multisite phosphorylation in interphase

(Kochin et al., 2014). Regardless, LMNA was similarly low and

decreased at the nuclear envelope (Figure 6E-i). Phospho-signal

normalized to total LMNA (pSer per LMNA) was also 2- to 3-fold

higher in CMs on soft gels than in stiff gels (Figures 6E-ii and 6E-

iii). blebb-treated cells on stiff gels again showed increased

pSer/LMNA for both phospho-sites. Signal in interphase cells

was, however, >10-fold lower than in rare mitotic CMs (Fig-

ure 6E-ii, inset: purple arrow). Studies of stable phospho-

mimetic mutants (GFP-S22A and GFP-S22E) conformed to the

model, with higher nucleoplasmic signal but lower overall levels

for S22E (Figures 6F-i and S7A). Inhibition of translation for 3 h

(Trx-i) showed more degradation only for S22E (Figures 6F-i

and S7B), and MMP2-i treatment increased S22E intensity (Fig-

ure 7C) consistent with inhibition of phospho-LMNA turnover.

S22E-phosphomimetic cells also exhibited higher DNA damage

(Figures 6F-ii and 6F-iii) and more cells in late cell cycle (4 N; Fig-

ure 6F-iv) that were more suppressed by acute blebb than S22A

cells. Reduction of nuclear stress by soft matrix or actomyosin

inhibition at the single-cell level thus favors LMNA interphase

phosphorylation, nucleoplasmic solubilization, and subsequent

degradation—while also suppressing DNA damage and cell-

cycle arrest.

DISCUSSION

LMNA defects cause disease through ‘‘cell-extrinsic mecha-

nisms’’ that likely include ECM and/or cytoskeletal stress.

Mosaic mice in which 50% of the cells express defective

LMNA maintain a normal lifespan, whereas mice with 100%

defective cells die within weeks of birth (de La Rosa et al.,

2013). Cultures on rigid plastic of the same cells (and similar

cell types [Hernandez et al., 2010]) exhibit premature senes-

cence and apoptosis, as is common with excess DNA damage,

but growth and viability are surprisingly rescued upon culture

on almost any type of ECM. Soft matrix certainly reduces

cytoskeletal stress and suppresses nuclear rupture and DNA

damage in CMs with low LMNA (Figure 3E). The findings

are further consistent with the observation that laminopathies

spare soft tissues such as brain, independent of lineage

or developmental origin, but generally affect stiff and mechan-

ically stressed adult tissues including muscle or bone (Cho

et al., 2018).

Early embryos are soft, with minimal ECM, but as tissues form

and sustain higher physical stresses, collagen-I accumulates,

and stiffening of the developing tissue minimizes the strain on

resident cells. However, increasing stiffness and stress necessi-

tate an adaptive mechanism to protect against nuclear stress,

and accumulation of mechanosensitive LMNA fulfills this role—

unless actomyosin stress is inhibited (Figures 1A–1C). LMNA

thus mechano-protects DNA by retaining repair factors in the nu-cleus (Figures 3A–3D), which thereby prevents excessive DNA damage in stiff microenvironments and/or under high actomy-

osin stress (Figure 7A). Rapid change in LMNA protein indepen-dent of any transcription or translation (Trx-i) rules out many possible contributing pathways to the equally rapid DNA dam-

age response. Such conclusions about causality are otherwise difficult to achieve with experiments conducted over many hours or days such as with mouse models. Results with the cardiac myosin-II specific inhibitor (MYK) in clinical trials for some car-diomyopathies (National Institutes of Health, 2016a [NCT02842242]) further suggest an unconventional method to attenuate DNA damage in the heart (Figures 1C and 1D).Loss of DNA repair factors could compromise genome integ-

rity, but enhanced entry of cytoplasmic nucleases (Maciejowski et al., 2015) would seem unlikely to generate gH2AX foci distrib-uted throughout the nucleoplasm (Figures 3E-ii, 4A-i). Indeed, cytoplasmic proteins that bind DNA strongly (e.g., cGAS) are restricted to rupture sites. Rupture results in ‘‘global’’ mis-localization of multiple DNA repair factors in several path-ways (Figure S2E-i) (Xia et al., 2018; Irianto et al., 2017), and repair factor depletion causes excess DNA damage before, during, and after transiently induced damage (by 1 h etoposide; Figure 4B).Remarkably, MMPs that degrade and remodel collagen-I

matrix within hours or less are found here to directly regulate LMNA of the ‘‘nuclear matrix’’ (Figures 5D-i, 6C, and 6D), which indicates a surprising inside-outside symmetry (Figure 7) to the ‘‘use it or lose it’’ mechanism (Figures 5C and 7B) of stress-sta-bilizing fibers. While both phospho-LMNA and MMP2 are found mostly in the nucleoplasm of CMs, the specific functions of MMP2 in the nucleus, the location of LMNA degradation, and its activation and localization mechanisms remain to be elucidated. Nuclear MMPs are not new (Xie et al., 2017), but tension regulation of their substrates has not been described. Our studies of isolated CMs on soft or stiff gels further demon-strate steady-state LMNA levels are determined primarily by a basal tone related to average morphologies of cells and nuclei, as opposed to dynamic contractions that intermittently strain the nucleus (Figures 3F, 6D, and S6G). Basal and dynamic strain might contribute to a ‘‘tension-time integral’’ model that seems predictive for hypertrophic versus dilated cardiomy-

opathy disease fates (Davis et al., 2016). Roles for MMPs in such diseases and compensatory changes in LMNA could emerge.Reasons why cells do not simply maintain high LMNA levels at

all times in all tissues could be physical as well as biochemical. An overly rigid nucleus might disrupt cytoskeletal assembly and function. Also, LMNA fine tunes differentiation and matura-tion by regulating many cytoskeletal genes via the MKL1/SRF pathway and by interacting with regulatory factors at the nuclear periphery. Nonetheless, accumulation of LMNA in stiff tissues of the embryo begins earlier than reported (Stewart and Burke, 1987) and tracks nonlinearly what eventually becomes the most abundant protein in adult animals, collagen-I (Figure 5C). Collagenase softens tissue and decreases LMNA consistent with scaling trends, and the scaling LMNA � collagen-I0.3 in em-

bryos matches meta-analyses of all available transcriptomes analyzed for diverse normal and diseased adult and developing


from Swift et al., 2013; Rober et al., 1989). Mechano-coupled

accumulation of LMNA and collagens that begin in early devel-

opment could thus persist well into maturation and perhaps

even to disease and aging of adult tissues (Figure 7G). Thus, me-

chano-protection of the genome by LMNA likely plays a critical

role not only in embryonic development but also in a broad range

of adult diseases.

A

B

‘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 fromDingal et al. (Dingal and Discher, 2014), with (ii) governing equations. Squares, genes; circles, protein. LMNA andmyosin-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 is consistent with that across proteomes of diverse chick embryo tissues at E18 (Fig. S7E), as well as adult mouse tissues (a = 0.4)(Swift et al., 2013), and underscores a universality of normal regulation. Even the tissue-dependent timing of LMNA detection (Solovei et al., 2013; Ro ber et al., 1989; Lehner et al., 1987) cor-relates well with stiffness of a tissue in adults (Fig. S7F; adapted


STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Embryonic Chick Hearts and Cardiomyocytes

B Induced Pluripotent Stem Cell-Derived Cardiomyo-

cytes (iPS-CMs)

B Cell Lines

d METHOD DETAILS

B Mass Spectrometry (LC-MS/MS) of Whole Heart

Lysates

B Immunoblotting

B Ex Vivo Drug Perturbations of Embryonic Hearts

B Morpholino Knockdown in Intact Hearts

B Alkaline Comet Assay

B Immunofluorescence Imaging

B siRNA Knockdown and GFP-Repair Factor Rescue

B mCherry D450A FOK1 TRF1 Expression

B Quantitative Fluorescence In Situ Hybridization and

Telomere Analysis

d QUANTIFICATION AND STATISTICAL ANALYSIS

B Quantification of Beating Strain

B Statistics

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.

devcel.2019.04.020.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health/National Heart

Lung and Blood Institute awards R01 HL124106 and R21 HL128187, National

Cancer Institute PSOC award U54 CA193417, the US-Israel Binational Sci-

ence Foundation, Charles Kaufman Foundation award KA2015-79197, and

National Science Foundation grant agreement CMMI 15-48571.

AUTHOR CONTRIBUTIONS

Conceptualization, S.C. and D.E.D.; Investigation, S.C., M.V., A.A., S.M., K.V.,

I.I., J.I., and M.T.; Validation, Y.X. and K.Z.; Formal Analysis, S.C. and D.E.D.;

Resources, E.T., F.M., H.T., R.G., and B.P.; Writing, S.C. and D.E.D.; Supervi-

sion, D.E.D.; Funding Acquisition, D.E.D.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: November 20, 2018

Revised: February 20, 2019

Accepted: April 16, 2019

Published: May 16, 2019

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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

(Ser139), clone JBW301

EMD Millipore Cat# 05-636; RRID: AB_2755003

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

cardiomyocytes (iPS-CMs)

Stanford Cardiovascular Institute

(SCVI) Biobank

http://med.stanford.edu/scvibiobank.html

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

mCherry–TRF1

Laboratory of Roger Greenberg,

Univ. of Pennsylvania

N/A

Experimental Models: Organisms/Strains

White Leghorn chicken eggs:Specific Pathogen

Free (SPF) fertilized

Charles River Laboratories Premium #10100326

Oligonucleotides

Vivo-Morpholino against chick LMNA (MOLMNA):

50-TTGGCATTCCTGCAACGCCGC-30Gene Tools N/A

50 mismatch control Vivo-Morpholino (MOCtrl):

50-TTCGCATTGCTGGAACGGCCC-30Gene Tools N/A

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 CardiomyocytesWhite 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 con-

taining PBS on a 37�C-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 vitellinemembrane 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 37�C in pre-

warmed chick heart media (a-MEM supplemented with 10 % FBS and 1% Penn-strep, Gibco, #12571) for at least 1 hrs for stabili-

zation, 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 37�C 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 37�C. 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 CMswere imaged using an Olympus I81microscope 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 4�C DMEM/F12 medium 1:200 dilution

(DMEM/F12medium #10-092-CM-Fisher), mixed gently, and 1ml of this suspension was added to one 6-well plate (Corning Catalog

#353046) and incubated for 1 hr at RT to allowMatrigel 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 mMof 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 LinesA549 (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 LysatesMass spectrometry (LC-MS/MS, or ‘MS’) samples were prepared using procedures outlined in Swift et al.. Briefly, �1 mm3 gel sec-

tions 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 37�C. 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 37�C with gentle shaking. The re-

sulting 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.

ImmunoblottingHearts 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 80�C for 10 min and centrifuged at maximum speed for 30 min at 4�C. 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), washed33 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 concen-

trations generally on the order of�1 mg/ml at 4�C overnight. After washing33 with TTBS, the membrane was incubated with 1:2000

diluted secondary Ab: anti-mouse HRP-conjugated IgG (GE Healthcare, Little Chalfont, UK), at RT forR1.5 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 HeartsFor 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 37�C, for the desired duration. Drug solutions were kept away from light to mini-

mize 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 experi-

ments. 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 37�C. 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 mMmifepristone (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 HeartsVivo-Morpholinos (MO) against LMNA mRNA and the corresponding 50-mispair control were designed according to the manufac-

turer’s guidelines (Gene Tools, Inc.): 50-TTGGCATTCCTGCAACGCCGC-30 (MOLMNA) and 50-TTCGCATTGCTGGAACGGCCC-30

(MOCtrl). Desired concentrations (1, 2, 5, 10, 30 mM) were prepared by dilution in heart growth medium at 37�C, and the resulting so-

lutions were added to E4 hearts for 24 hrs before analysis.


Alkaline Comet AssayAlkaline comet assays was performed according to manufacturer’s protocol (Cell Biolabs). Briefly, cells were trypsinized, mixed with

liquefied agarose at 37�C, placed drop-wise onto the supplied glass slide, and incubated for 15 min at 4�C 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 300mA for 30min 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 ImagingCells were rinsed with prewarmed PBS, fixed with 4% paraformaldehyde (PFA, Fisher) for 15min, washed33 with PBS and permea-

bilized with 0.5% Triton-X (Fisher) in PBS for 10 min. Permeabilized cells were then blocked with 5% BSA in PBS forR1.5 hrs. Sam-

ples were incubated overnight with primary antibodies in 0.5% BSA solution, with gentle agitation at 4�C. 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 weremountedwithmountingmedia (Invitrogen ProLongGold 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 45min, washed33with PBS, and permeabilizedwith 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 4�C (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 RescueAll 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 ExpressionDeath 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 AnalysisIsolated 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 telo-

mere 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 StrainTransfected 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 mmof each other were selected as fiduciary markers along the outer walls of ventricular tissue. A customMATLAB 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 pro-

jected 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.

StatisticsAll 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 per-

formed using the Kolmogorov-Smirnov test, with a = 0.05. Information regarding statistical analyses are included in the figure leg-

ends. 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).


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, KennethVogel, Yuntao Xia, Irena L. Ivanovska, Jerome Irianto, Manorama Tewari, KuangzhengZhu, 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

Page 1 of 5

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 2 of 5

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 3 of 5

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 4 of 5

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 5 of 5

(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)).

Figure S1

Myosin-II inhibition RescueD i

ii

LMNAH2AX

M: 10 2 10 30 10 (48h)

MOLMNAMOC rl

LMNA RA AGN

73HSP90 LMNA

RA AGN7390

H2AX 42

17

F G

H2AX 17

42

i ii

0 3 72

LMNA

E

0 00

0 02

0 04

0 06MOLMNAMOC rl

**+Bl bb

***B

r

AR /

ARr

f

0 10 20 300

50

100

***+Bl bb

MO r ( M)

b /

(BPM

)

0 10 20 300 0

0 5

1 0

1 5

2 0LMNAH2AX

**

***

MOC rl

MO r ( M)

Dry

r

5'

h

MO

Crl

(AU

)

i ii

T r A A A

A B

MSTPSQRRSG RGGGPSGTPL SPTRITR LAVYIDKVRL RITESEEVVS REVSGIK KT LDSVAKERAR

VRE EHKELKARNA KKR DLRAQVAK LEGALSEAKK RV DAENRLQTLK

EELEFQK ETKRR HETR QQEFESK RQHEDQIRHYRD ELEKTYGAKL ENAKQSAER

QLAAKEAKLR ER EGGRRLLAEK EREMAEMRARLALDM EINAYRKLLE GEEERLRLSP SPSSQRGARS

SGLQHSGAGS AKKRRLEDGE GREGREGRTS FSHHARTSGR FVRLRNKSN EDQALGNWQV KRQNGDDPPL TYRFPPKFTL

KLVRTVIINDDDEDEEDD EVSIHHR SR

G YR

K

LQE KEDLQELNDR SLELENAGLR AAY EAELADARLQLELSK EADLLAAQ ARLKDLEALL NSKEAALSTALGEK NLENE VR QLQDEMLR

NIY SEELR LVEIDN GR L AEALQDLRN SSMAGAAHEE LQQTHIRIDS

LSAELSQLQK EVEEALSRMQQQLDEYQE LLDIK

VGVEEVDLEGR AGQAVTIWASGAGATHSPP SDVVWKAQSS WGSGDSLRTA LINSNGEEVA MR

HHH SGCSGSADPA EYNLR TVL CGTCGQPADKGSAAAASSAS SASTVTVSR SSGGGIGE GLLGRSYVLG GAGPRRQAPAPQGCSIM

S q v r =

51

101

151

201

251

301

351

401

451

501

551

601

651

1

T

EERLRLSP SPSSQRGARSSGLQHSGAGS AKKRRLEDGE GREGREGRTS FSHHARTSGR VGVEEVDLEGFVRLRNKSN EDQALGNWQV KRQNGDDPPL TYRFPPKFTL KR AGQAVTIWA

KLVRTVIISGAGATHSPP SDVVWKAQSS WGSGDSLRTA LINSNGEEVA MRNDDDEDEEDD EVSIHHR SRHHH SGCSGSADPA EYNLR TVL CGTCGQPADK

G YRGSAAAASSAS SASTVTVSR SSGGGIGE GLLGRSYVLG GAGPRRQAPAPQGCSIM

0

20

40

60

80

100

73 42 17

DMSO (0 5h)Bl bb (0 5h)Bl bb (4 5h)W h (+1h)LMNA

H2AX

MW r (kD )

blD

ry (A

U)

DMSO (05h

)

Bl bb (0

5h)

Bl bb (1

h)

Bl bb (2

h)

Bl bb (3

h)

Bl bb (4

5h)

Wh

(+1h

)

Wh

(+3h

)

DMSO (6h)

+ OM (7

5h)

0 0

0 5

1 0

1 5

C b

rll

bl

Tl

y (A

U)

LMNA

HSP90 DMSO

90

73

80

60

50

40

30

20

15

110

160

LMNB1 (

21)

LMNB2 (

28)

GAPDH (15)

HSP70 (3

5)

bl

(12)

(10)

0 0

0 5

1 0

1 5

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Housekeeping proteins

Prot (#

pept)

M S

y

Nr

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MSO

A & DNA’

U r RA AGN0 0

0 5

1 0

1 5 L AH2AX

blr

r (3

h)

U r RA AGN0 0

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1 0

1 5

2 0

**

blr

r (7

2h)

0 1 2 3 4 5 6 7 80

20

40

60

80

100

W h

+OM

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b /

(BPM

)

0 00

0 02

0 04

0 06Bl bb (25 M)

W h

DMSO C rlMYK (1 M)

+OM

T b

r

AR /

ARr

f

C

LMNA

H2AX

80

60

50

40

30

20

15

80

60

50

40

30

20

15

110

160

DMSO (05h

)

Bl bb (0

5h)

Bl bb (1

h)

Bl bb (2

h)

Bl bb (3

h)

Bl bb (4

5h)

Wh

(+1h

)

Wh

(+3h

)

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+ OM (7

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iii

i

ii0

50

100

150

90 kD 73 kD

HSP90LMNA

blD

ry (A

U)

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Bl bb (1

h)

Bl bb +

Tr (1

h)0 0

0 5

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rll

bl

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y (A

U)

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90

100 0

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1 0

1 5

2 0

70 75

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72T (h)

AR /

ARr

fr

DM

SO

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v H

SP90

)N

r

Ur

0 1 2 3 4

1

1 2 3 4 50

Day 3 (post-treatment)Day 0

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AR /

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0 5 100 4 1 100 5 2 100 5 2 100 5 3 100 50

5 0 104

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1 5 105

2 0 105

AGNRAU r 0 9988

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R2

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17

42

73

Figure S2

A

F

D

NT C l LMNA LMNA0

2

4

6

8

***

+Bl bb

KU

80 N

l/C

yl

i ii

Ex vivo

53BP1 RPA2 DNA

Cytoplasmic

0 20 40 60 800 0

0 5

1 034

D (μ )

Rl

y (A

U)

B

0 2 40

20

40

60

24

+

T (h)

rf

ll

/ fr

DN

A

0 2 4 24

Rl

v

y (A

U)

Cytopl.

0 0

0 5

1 0B f rAf r

0 20 400 0

0 5

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D (μ )

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0 2 4 6 8 100 8

1 0

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0 8

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y

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/Ar

rf

NT C l LMNA LMNA0

5000

10000

+Bl bb

Tl K

U80

y

(AU

)

0 4 0 6 0 8 1 0 1 212

14

16

18

20

NTC l

LMNA

LMNA + Bl bb

LMNA + OM

R2 = 0 9145r = 0 9563

K 80 N l /Cy l

γH2A

X f

#

ii

iE

i ii

DMSOBl bb

Wh

Wh +

Wh +

Mf

r

Wh +

0

10

20

**

*

(KU80

NLS

)

ll

/ K

U80

ll

G0 24

Figure S3

0 5000 10000 15000 200000

20

40

60

80C lK 80

KU80 l y (A U )

γH2A

X f

#0

50

C l2N = 92 4N = 8

0

50

LMNA2N = 84 4N = 16

0

50

KU802N = 85 4N = 15

0

50

BRCA12N = 78 4N = 22

0

50

RPA12N = 81 4N = 19

1 2 40

50

C b2N = 59 4N = 41

N r l DNA (A U )

f

l

C l LMNA KU80 BRCA1 RPA1 C bC lLMNA ****KU80 **** ****BRCA1 **** ****RPA1 **** **** ** *C b **** **** *** ***

A

D

i ii

GFP:GFP KU70

G

LMNALMNB2KU80RPA1

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253

193

88

Ei ii

iii

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0 1 2 3 41

10

100

1000

10000

T l DNA (A U )

U

y (A

U)

G2G1

S

G1 S G20

20

40

60

80 WTLMNALMNA + Bl bb

****

f

l

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Pll

l

GFPl

GP

ll

l

GFPl

GP

ll

l

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G

107

108

109

101 0

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KU70 KU80 LMNA

52

2

7

MS

y

(AU

)

C C b0

5

10

15

20 DMSOBl bb

γH2A

X f

C rl LMNA0

1

22N4N

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***γH2A

X f

r

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A (A

U)

C

Figure S4

C

1 10108

109

R2 = 0.9963

Lamin-A ~ Et0.750

4

610

18*

T ff E (kP )

LA

M S

LFQ

y

106 107 108 1091

10

Et ~ <Collagen-I> 0.52

R2 = 0.9975

4

6

10

18*

0 4 α 0 65f r R2 > 0 95

C llM LFQ y

T

ffE

(kP

)

107

108

109

101 0

101 1

4 618*

10

R2 = 0.9687

1

Lamin-B1 ~ Const.Lamin-B2 ~ E 0.25

<Lamin-B>

10

T ff E (kP )

LB

M S

LFQ

y

E F

A B

Lamin-A

HSP90 84

73

68

D

0 100 200 3000

1 0×101 0

2 0×101 0

3 0×101 0

4 0×101 0

R l C l1 1R l C l1 2

R2=0 9995

C ll >

C ll k (μ )

M S

MS

y

(AU

)

Br H r L v r0

2

4

6

*

10

ff

E (k

P)

h

125 8151 65132 32137 538 64296 08178 137324 63273 25168 5151 96963 696339 59

COL1A2VTN

FBLN2COL1A1

LUMPOSTNFBLN1FBN2/3

FN1LAMB1MFAP1

LNCOL6A3

41 79242 019226 5221 8283 305223 6229 04182 15140 79102 83101 49231 9964 09185 113222 81104 21

611172726827259

PXNVCL

TLN1RDXTLN2

ZYX

ACTBACTC1

MYH9MYH7

ABL M1MYH15MYH10

MYOM1MYBPC1

ACTN2ACTN1

MYO18APDL M5

L MA1MYH3

ACTN4

0 1 10

LMNALMNB1LMNB2

736768

G

0 2 4 6 8 10 12 140

2

3

4

1

* LMNA

LMNB

bry ( y )

blr

4 (A

U)

iii

4 60

1

2

LA

:B r

by

F (A

U)

Figure S5

B

MMP2

DMSO Bl bb C l'

+ Bl bb

6875

42

DMSO Bl bb C l' Tr MMP CDK0 0

0 5

1 0

1 5

+ Bl bb

Cl

v/P

rM

MP2

by

bl (A

U)

0 0

0 5

1 0

1 5

RP

A1

MS

y

(AU

)

DMSO Bl bb C l' Tr MMP CDK0 0

0 5

1 0

1 5

2 0

+ Bl bb

Pr

MM

P2

MS

y (A

U)

LMNA

LMNA

LMNAfr

T

ALMNA

HSP90

73

73

84

73

-- + + + + +

1

*****

*

0 5LMN

A (A

U)

-- + + + + +

S r22 / LMNA

1*

***

* *

*

3

S r390 / LMNA

0 5

** *

*

Ph

hry

lLM

NA

(AU

)

C

i

ii

iii

A B

D

0 1 1 10 1001

2

3

4

5

106 107

Rigidplastic

*** *

**

46

10

in vivo ( 4 10)in vitro 4 CM

G l S ff E l (kP )

Cll A

R (

lr

)

Ci

ii

F G Hi

ii

0 1 1 10 1000 00

0 01

106 107

0 15

0 20

0 25

0 30

0 35

Rigidplastic

***

*

**

4

6

10

Bl bbDMSO C rlBl bb + GMBl bb + ARPin vivo ( 4 10)

in vitro4 CM

G l S ff E l (kP )

Nl/

Cy

r

fr

0 5 10 150

100

200

250

300

350

*

25

bry ( y )

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r vl

( μ3 )

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2

4

6

*

bry ( y )

Nl

j

r

l1

/ ff

f v

r (C

OV

)

0

30

6090

120

150

180

210

240270

300

330

0

Embryonic heart stiffness

0 1 1 10 1000

5

10

15

20

25

*

+Bl bb

G l ff E l (kP )

b

ll

0 1 1 10 1000 00

0 05

0 10

0 15***

* C llN l

+Bl bbNr

b

rΔA

R /

AR

rf

0 1 1 10 1000 00

0 02

0 04

0 06 *

+Bl bbCll Δ

Ar

/ A

rr

f

i

ii

iii

5 10 15

108

TUBA1C (45 9 kD )TUBB7 (49 67 kD )TTLL12 (72 81 kD )

1.7x0.5x

1.7x

bry ( y )

Tb

l

TTL

MS

y

(AU

)

Figure S6

DMSO OM MYK0

1

2

*

Rigid plastic

LA

:B r

(AU

)

0 00 0 05 0 10 0 150 00

0 04

0 08

0 12

0 3 kP

1 kP

10 kP34 kP

r = 0 9097R2 = 0 8276

Sl 0 7

C ll ΔAR/ ARr f

Nl

ΔAR

/ A

Rr

f

0 00 0 05 0 10 0 150 0

0 5

1 0

1 5

0 3 kP

1 kP10 kP

34 kP

r = 0 2199R2 = 0 04837

C ll ΔAR / ARr f

LA

:B r

(AU

)

in vivo

anti-GFP

Chicken E18 embryo

E18 HeartsE F

4

4

5

C l1 2

LL

L b1 L b2>

M h r v lL l Physiol Genomics 2014

18 57 5 bry

l 2(Col1a1)

l2(

mR

NA

)

Col1a2 Lo

xLo

xl1Lo

xl2Lo

xl3Lo

xl4Tgm

2Mmp1

Mmp8

Mmp13Mmp2

Mmp9Acta

2Myh

9

Tuba1

c TtlSun

1Sun

2Emd

Lmna

Lmnb

1

Lmnb

2Xrcc

6RaraRarbRargYap

1Gata

4

Nkx2.5

0 0

0 5

1 0mRNA ~ Col1αmRNA

C l X l k r MMP N L Tr rf r

Crr

l v

ff

*******

***

**

**

***

***

Cy k l

Av

r

l

αm

RN

A >

vC

ol1a

1

5 0 5 10 15 205

10

15

20

25

30

M hy

L

K yL v r

Br

R2=0.8673

R b r l Development 1989

H r

S l v l Cell 2013

CM

A fr S f l Science 2013T ff E (kP )

Dbl

r

f lA

(br

y

y)

108 109

108

109

Br

B r f brH r

K y

L v r

L

Br l

S l

T

Ov r

C l1 2L

Lamin-A notdetected in brain

U bb lMol Biol Evol 2015

C l1 1MS LFQ y

MS

LFQ

y

109

108

109

F1M2

F2

M3

F3

M4

M5F5

R2= 0 8547R2= 0 9967

C l1 1

Nr

MS

y

+ DMSO +

GF

GF

BA

i

ii iii

0 5 10 150 0

0 5

1 0

1 5 GFP S22AGFP S22A + TrGFP S22GFP S22 + Tr

D (μ )

Nr

GFP

y

(AU

)

GFPS22

A

GFPS22

A + Tr

GFPS22

GFPS22

+ Tr

0 0

0 5

1 0

1 5 ***

Nl

l/L

r LMNC

LMNA

β

i

ii

C

GFP S22 GFP S220

1000

2000

3000

4000

+ MMP2

*

LA

/CR

l y

(AU

)

0 2 40 0

0 5

1 0

1 5

2 0

24

GFP S22AGFP S22A + TrGFP S22GFP S22 + Tr

T (h)

Nr

GFP

y

(AU

)

80

60

50

40

30

20

110

160

15

GFPS22

A

GFPS22

A + Tr

GFPS22

GFPS22

+ Tr

LMNALMNC

LMNA/C

0 0

0 2

0 4

0 6

0 8

1 0

1 2

6080110 50 40 30 20 15

GFP S22A + TrGFP S22A

GFP S22GFP S22 + Tr

Fr

M l l r h (kD )L

A/C

ry

r

β (A

U)

D

Figure S7

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]://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 regu-lated in response to mechanical stimuli to prevent the nucleus from rupture, keep DNA repair factors in thenucleus, and consequentially ‘‘mechano-protect the genome.’’

The cardiac microenvironment constantly

changes during development and dis-

ease, and the associated mechanical

properties affect cellular responses,

such as proliferation or differentiation

(Ward and Iskratsch, 2019). The stiffness

of the microenvironment is sensed by

the resident cells either through cell-sur-

face receptors and attached adaptor

proteins, such as talin, or directly by the

nucleus (Pandey et al., 2018; Swift et al.,

2013). Previous work from the Discher

lab demonstrated that the nuclear enve-

lope composition and especially the

lamin-A (LMNA):lamin-B ratio is not only

regulated downstream of mechanical sig-

nals, but can also modify differentiation

pathways (Swift et al., 2013). Moreover,

recent work from migratory cells high-

lighted how nuclear lamina composition

(and stiffness) can affect nuclear defor-

mation and lead to DNA damage, thus

linking mechanical signaling to genomic

instability (Denais et al., 2016; Raab

et al., 2016). However, while previous

studies suggested that DNA damage

can be a factor leading to heart failure

(Higo et al., 2017), how stiffness, nuclear

mechanics, and DNA integrity are all

associated in the context of heart devel-

opment or disease remained unresolved.

In the current issue of Developmental

Cell, Cho et al. (2019) use a wide range

of techniques and experimental models

to analyze the role of LMNA in cardiomyo-

cyte mechanosensing.

The authors initially set out to study the

interference of cardiomyocyte contrac-

tility and collagen matrix rigidity in embry-

onic chick hearts, using pharmacological

inhibitors, collagenase, or transglutami-

nase. The authors detected significant

effects on LMNA levels and concurrent

DNA damage, suggesting a link between

mechanical forces and genome integrity

in the heart. This hypothesis was sup-

ported by LMNA knockdown, which led

to nuclear rupture and increased DNA

damage. To further explore the link be-

tween extracellular matrix (ECM) stiffness,

nuclear integrity, and DNA damage, the

authors first knocked down LMNA in

embryonic hearts. This led to the mis-

localization of fluorescently tagged DNA

repair factors outside of the nucleus.

Further, when human induced pluripotent

stem cell-derived cardiomyocytes (hIPS-

CM) were cultured on either stiff or soft

matrices, DNA repair factors were again

mis-localized on stiff surfaces due to

nuclear envelope rupture, which was

aggravated by LMNA knockdown. Im-

portantly, in both scenarios the effects

were blocked by simultaneous blebbista-

tin treatment, underlining the role of

contractile forces in this process. Last,

cyclic (passive) stretching of cardiomyo-

cytes and U2OS cells also led to DNA

damage and mis-localization of DNA

repair factors especially after LMNA

knockdown, confirming the link between

mechanical forces, LMNA levels, and

nuclear damage.

To study how LMNA levels were

regulated downstream of mechanical

signaling, a range of mass spectrometry

(MS) analyses was performed. The au-

thors first confirmed previous results

showing that LMNA levels and collagen-I

are tightly linked to the changing stiffness

of the heart during development. Next, the

authors profiled the proteome of embry-

onic hearts after acute (1 h) drug treat-

ments. Levels of collagen-I, LMNA, and

vinculin were decreased after blebbistatin

treatment, but LMNA and collagen-I were

rescued after simultaneously treating with

a matrix metalloproteinase (MMP) inhibi-

tor, suggesting that MMPs can degrade

both collagen in the ECM and LMNA in

the nucleus. Intriguingly, the simultaneous

treatment of cells with a cyclin-dependent

Developmental Cell

kinase (CDK) inhibitor and blebbistatin

also resulted in a rescue of LMNA levels,

implying a role for CDKs as regulators of

LMNA turnover. CDKs were previously

shown by the authors to phosphorylate

LMNA, which regulated the targeting of

LMNA from the nuclear lamina to the

nucleoplasm and its subsequent proteo-

lytic degradation (Buxboim et al., 2014).

Consistently, the authors detected an in-

crease in phospho-LMNA fragments after

blebbistatin treatment, but a reduction

after simultaneous treatment with a pan-

MMP or a MMP2 inhibitor.

These experiments linked CDKs to

proteolytic degradation of LMNA by

MMP2 and also to the regulation of nu-

clear stiffness and DNA integrity. Im-

portantly, immunoprecipitation combined

with MS additionally indicated that repair

factors interacted preferentially with

non-phosphorylated LMNA compared to

its phospho-solubilized nucleoplasmic

form, suggesting that LMNA in its unphos-

phorylated nuclear lamina-associated

form also acts as scaffold for the repair

factors.

This involvement of CDKs in stiffness-

dependent phosphorylation of LMNA is

intriguing in several regards. First, CDKs

were previously shown to affect the

localization of other nuclear envelope pro-

teins, such as SUN, KASH5, and lamin-

C2; however, the mechanism remained

elusive (Viera et al., 2015). Could it be sec-

ondary to the loss of LMNA or are these

regulated in a similar manner? Second,

levels of certain endogenous CDK inhibi-

tors (especially p21 and p27) were previ-

ously found to increase during heart

development, but significantly decreased

in heart disease (Burton et al., 1999). This

could suggest that LMNA degradation

due to its phosphorylation by CDKs con-

tributes significantly to disease progres-

sion and heart failure. Finally, the findings

49, June 17, 2019 ª 2019 Elsevier Inc. 821

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https://doi.org/10.1016/j.devcel.2019.05.041

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Embryonic Heart Healthy Adult Heart

Integrins Talin Vinculin LMNAECM (Col1 etc.)

CDKCDKi

Cyclin

Diseased Adult Heart

CDKCyclin

Stiff NucleusNo Rupture H

igh

Act

omyo

sin

Con

tract

ility

Hig

h A

ctom

yosi

n C

ontra

ctili

ty

NUCLEARRUPTURESoft NucleusLo

w A

ctom

yosi

n C

ontra

ctili

ty

P

CDKCyclin

Soft Nucleus No Rupture

P

MMP2

PP P

MMP2

P

DNA Repair Factors

DNA Repair Factors Sarcomeres

Figure 1. Nuclear Mechanosensing through LMNA and Potential Implications in Heart DiseaseThe stiffness of the ECM changes during development. Simultaneous increases in LMNA in the nuclear lamina, due to changes in expression and turnover, stiffenthe nucleus and prevent nuclear rupture. Endogenous CDK inhibitors are strongly reduced in heart disease, leading to higher CDK activity, removal of LMNA fromthe nuclear lamina, and its degradation in the nucleoplasm by MMP2. The softer nucleus is unable to resist the contractile forces, leading to nuclear rupture andloss of DNA repair factors, which are otherwise associated with LMNA.

Developmental Cell

Previews

by Cho et al. could also have implications

for cancer therapy and explain the cardio-

toxicity of doxorubicin and perhaps other

chemotherapeutic agents, since CDK ac-

tivity in the heart was increased due to

doxorubicin chemotherapy (Xia et al.,

2018). According to Cho et al., this would

lead to increased phosphorylation and

degradation of LMNA, hence a softer nu-

cleus, more nuclear rupture, and DNA

damage, possibly explaining the link to

heart failure (Xia et al., 2018). Indeed,

treatment with CDK inhibitors preserved

the heart function.

In summary, Cho et al. provide strong

evidence that LMNA expression and turn-

over depends on ECM stiffness and is

regulated by CDKs and MMP2 (Figure 1).

If forces are low, LMNA becomes phos-

phorylated by CDKs, then moves to the

nucleoplasm and is degraded by MMP2.

In embryonic hearts, where the ECM is

soft, LMNA is present at low levels in

part due to changes in expression

(through self-regulation via the retinoic

acid pathway [Swift et al., 2013]), but

presumably also in part because of CDK

activity, consecutive removal from the nu-

clear envelope, and subsequent degrada-

tion. On the contrary, in adult hearts,

where the matrix is stiffer, LMNA is stabi-

lized in the membrane in order to prevent

nuclear rupture and DNA damage, in what

the authors call a ‘‘lose it or use it’’ mech-

822 Developmental Cell 49, June 17, 2019

anism. How CDKs or MMP2 are regulated

through cytoskeletal contractility or stiff-

ness, however, remains unresolved. The

response to blebbistatin is fast, thus

excluding transcriptional regulation and

rather suggesting a more direct mechani-

cal activation. Therefore, evidence points

to cardiomyocytes sensing matrix rigidity

through a network of mechanosensitive

proteins, including talin at integrin adhe-

sions and LMNA in the nuclear envelope.

However, the intermediate molecules

and interactions remain to be elucidated.

REFERENCES

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Ward, M., and Iskratsch, T. (2019). Mix and (mis-)match - The mechanosensing machinery inthe changing environment of the developing,healthy adult and diseased heart. Biochim.Biophys. Acta. Mol. Cell Res. Published onlineFebruary 8, 2019. https://doi.org/10.1016/j.bbamcr.2019.01.017.

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