10.1161/CIRCULATIONAHA.121.055313
Cardiovascular Progerin Suppression and lamin A Restoration Rescues
Hutchinson-Gilford Progeria Syndrome
Running title: Sánchez-López et al.; Cardiovascular Progerin Targeting Extends Lifespan
Amanda Sánchez-López, PhD1,2†; Carla Espinós-Estévez, MS1†;
Cristina González-Gómez BS1,2; Pilar Gonzalo, PhD1,2, María J. Andrés-Manzano1,2;
Víctor Fanjul, PhD1,2; Raquel Riquelme-Borja, MS1; Magda R. Hamczyk, PhD1,2††;
Álvaro Macías, PhD1,2; Lara del Campo, PhD1,2; Emilio Camafeita, PhD1,2;
Jesús Vázquez, PhD1,2; Anna Barkaway, PhD3; Loïc Rolas, PhD3; Sussan Nourshargh, PhD3;
Beatriz Dorado, PhD1,2; Ignacio Benedicto. PhD1; Vicente Andrés, PhD1,2,*
1
Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain;
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2
CIBER de Enfermedades Cardiovasculares (CIBERCV), Spain; 3Centre for Microvascular
Research, William Harvey Research Institute, Barts and The London School of Medicine
and Dentistry, Queen Mary University of London, London, UK
†
Co-first authors with equal contribution
Current address: Departamento de Bioquímica y Biología Molecular, Instituto Universitario de
Oncología (IUOPA), Universidad de Oviedo, 33006 Oviedo, Spain
††
Address of Correspondence:
Vicente Andrés
CNIC, Melchor Fernández Almagro 3, 28029
Madrid, Spain
Phone: +34-91 453 12 00 (Ext. 1502)
E-mail: vandres@cnic.es
*This article is published in its accepted form, it has not been copyedited and has not appeared in an
issue of the journal. Preparation for inclusion in an issue of Circulation involves copyediting,
typesetting, proofreading, and author review, which may lead to differences between this accepted
version of the manuscript and the final, published version.
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Abstract
Background: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by
premature aging and death mainly due to myocardial infarction, stroke, or heart failure. The
disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells.
Patients look healthy at birth, and symptoms typically emerge in the first or second year of life.
Assessing the reversibility of progerin-induced damage and the relative contribution of specific
cell types is critical to determining the potential benefits of late treatment and to developing new
therapies.
Methods: We used CRISPR/Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice
engineered to ubiquitously express progerin while lacking lamin A and allowing progerin
suppression and lamin A restoration in a time- and cell-type-specific manner upon Cre
recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with
Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A
ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells
(VSMCs) and cardiomyocytes.
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Results: Like HGPS patients, HGPSrev mice appear healthy at birth and progressively develop
HGPS symptoms, including failure to thrive, lipodystrophy, VSMC loss, vascular fibrosis,
electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26
months in wild-type controls, p<0.0001). Ubiquitous progerin suppression and lamin A restoration
significantly extended lifespan when induced in 6-month-old mildly symptomatic mice and even
in severely ill animals aged 13 months, although the benefit was much more pronounced upon
early intervention (84.5% lifespan extension in mildly symptomatic mice, p<0.0001, and 6.7% in
severely ill mice, p<0.01). Remarkably, major vascular alterations were prevented and lifespan
normalized in HGPSrev mice when progerin suppression and lamin A restoration were restricted
to VSMCs and cardiomyocytes.
Conclusions: HGPSrev mice constitute a new experimental model for advancing knowledge of
HGPS. Our findings suggest that it is never too late to treat HGPS, although benefit is much more
pronounced when progerin is targeted in mice with mild symptoms. Despite the broad expression
pattern of progerin and its deleterious effects in many organs, restricting its suppression to VSMCs
and cardiomyocytes is sufficient to prevent vascular disease and normalize lifespan.
Key Words: Hutchinson-Gilford progeria syndrome; Progerin; Vvascular Smooth Muscle Cell,
Cardiomyocyte
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Non-standard Abbreviations and Acronyms
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BSA
CNIC
ECG
h
H&E
HGPS
HGPSrev
HRP
IP
LC-MS/MS
min
MRI
NGS
OT
o/n
PRM
RT
SD
SEM
SEMS
SM22α-Cre
SMA
TBST-T
Ubc-CreERT2tg/+
VSMC
WT
Bovine serum albumin
Centro Nacional de Investigaciones Cardiovasculares
Electrocardiography
Hour(s)
Hematoxylin-eosin
Hutchinson-Gilford progeria syndrome
LmnaHGPSrev/HGPSrev mouse
Horseradish peroxidase
Immunoprecipitation
Liquid chromatography coupled to targeted tandem mass spectrometry
Minute(s)
Magnetic resonance imaging
Normal goat serum
Off-target
Overnight
Precursor-reaction monitoring
Room temperature
Standard deviation
Standard error of the mean
Spin-echo multi-slice
B6.Cg-Tg(Tagln-cre)1Her/J mouse
Smooth muscle α-actin
Tris-buffered saline supplemented with 0.2% Tween 20
B6.Cg Ndor1Tg(UBC-cre/ERT2)1Ejb/1J mouse
Vascular smooth muscle cell
Wild-type
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Clinical Perspective
What is new?
•
We have generated a new HGPS-like mouse model that ubiquitously expresses progerin,
lacks lamin A, and allows progerin suppression and lamin A restoration in a time- and
cell-type-specific manner upon Cre recombinase activation.
•
Progerin suppression and lamin A restoration extended lifespan in mice with mild
symptoms and even in severely ill animals, although the benefit was much more apparent
in mildly symptomatic animals.
•
Despite the broad expression pattern of progerin and its deleterious effects in many
organs, restricting its suppression and lamin A restoration to VSMCs and cardiomyocytes
prevented the development of vascular pathology and normalized lifespan in HGPSrev
mice.
What are the clinical implications?
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•
Our preclinical studies demonstrate that it is never too late to treat HGPS, although the
benefit was much more pronounced when progerin suppression and lamin A restoration
were achieved at early stages of disease progression.
•
Strategies to treat HGPS through gene therapy or RNA therapy should consider
targeting VSMCs and cardiomyocytes.
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Introduction
HGPS is an ultra-rare genetic disorder (estimated prevalence 1 in 18-20 million people;
https://www.progeriaresearch.org/) characterized by accelerated aging and premature death
(average lifespan: 14.6 years)1-3. Most HGPS patients are heterozygous for a de novo synonymous
mutation in the LMNA gene (c.1824C>T; p.G608G) that activates the use of a cryptic splice donor
site in exon 114, 5. In normal cells, LMNA expression mostly generates the alternatively-spliced
isoforms lamin A and lamin C. The HGPS-causing mutation creates an aberrant LMNA mRNA
that lacks 150 nucleotides in exon 11; this is translated into progerin, a permanently-farnesylated
lamin A variant that exerts a dominant-negative effect1. Growth failure and alopecia manifest in
HGPS patients as the first disease symptoms typically in the first or second year of life. Additional
symptoms develop and worsen over time, including dermal and bone abnormalities, joint
contractures, and loss of subcutaneous fat. The main medical problem in HGPS is severe
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cardiovascular disease, including generalized atherosclerosis and vascular calcification and
stiffness, which ultimately provoke myocardial infarction, stroke, or heart failure, the causes of
death in most HGPS patients6, 7. The US Food and Drug Administration recently approved the
treatment of HGPS patients with lonafarnib (marketed as Zokinvy™), a repurposed
farnesyltransferase inhibitor that has extended the lifespan of HGPS patients by 2.5 years (17%
increase)8-10. Nevertheless, there is a great need for better therapies to improve and eventually cure
HGPS.
HGPS patients are typically diagnosed when symptoms are present or even severe, and
treatment has historically been initiated at different disease stages6. It is therefore important to
ascertain how late in life treatment can be initiated in symptomatic individuals while still yielding
clinical benefit. Moreover, because progerin is expressed in most differentiated cells, it is vital to
identify the cell types that would most benefit from treatment. To address these questions, here we
generated the LmnaHGPSrev/HGPSrev (HGPSrev) mouse model, engineered to ubiquitously express
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progerin and lamin C and lack lamin A, while allowing progerin suppression and lamin A
restoration upon Cre recombinase activation. We have characterized this model to assess the
reversibility of progerin-induced damage by targeting progerin at early and late disease stages.
Moreover, we have examined the consequences of suppressing progerin and restoring lamin A
specifically in vascular smooth muscle cells (VSMCs) and cardiomyocytes, the major progerin
targets.
Methods
Additional methods are provided in the Supplemental Methods section online.
Data, analytical methods, and study materials will be made available to other researchers for the
purposes of reproducing these results or replicating these procedures upon reasonable request
directed to the authors’ laboratories.
Study approval and mouse models
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Mice used in this study were housed in the animal facilities at the Centro Nacional de
Investigaciones Cardiovasculares (CNIC) under specific pathogen-free conditions at a constant
temperature of 23°C, relative humidity 58%, and a 12-hour (h) dark/light cycle. Mouse health was
monitored in a blinded manner at regular intervals throughout the study. Mouse handling and
experimental procedures were performed to conform with current EU guidelines (Directive
2010/63/EU) and Recommendation 2007/526/EC regarding the protection of animals used for
scientific purposes, enforced in Spanish law under Real Decreto 1201/2005; all procedures were
approved by the Animal Protection Area of the Comunidad Autónoma de Madrid (PROEX
051/18) and the CNIC Ethics Review Board. To maximize information and minimize the number
of animals used, we followed the 3Rs principles11 and the ARRIVE guidelines12 throughout this
study.
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Studies were carried out in C57BL/6J mice fed a regular rodent chow diet. Equal numbers
of females and males were used. Ubc-CreERT2tg/+ mice (B6.Cg Ndor1Tg(UBC-cre/ERT2)1Ejb/1J)13 and
LmnaG609G/G609G mice14 were kindly provided by Dr. Mariano Barbacid and Dr. Carlos LópezOtín, respectively. The generation of HGPSrev mice is explained below. HGPSrev mice were
crossed with Ubc-CreERT2tg/+ mice to generate LmnaHGPSrev/HGPSrev Ubc-CreERT2tg/+ mice with the
Cre transgene in heterozygosis and exhibiting time-conditional Cre activity (referred to as
HGPSrev-Ubc-CreERT2 mice). SM22α-Cre mice (B6.Cg-Tg(Tagln-cre)1Her/J)15 (The Jackson
Laboratory, Bar Harbor, ME USA) were crossed with HGPSrev mice to generate
LmnaHGPSrev/HGPSrev-SM22α-Cre mice (HGPSrev-SM22α-Cre mice).
Double stranded (dsDNA) donor template design for LmnaHGPSrev strain generation
For homology directed repair, a 2,494 bp dsDNA donor template flanked by EcoRI and NotI
recognition sites was synthesized (Figure IA in the Supplement) and inserted into the pcDNA3.1
vector (Genscript; Piscataway, NJ USA). The dsDNA donor template contains a 938-bp left
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homology arm (Lmna intron 9, exon 10 and part of intron 10), a 672-bp insert harboring a loxPflanked cDNA containing exons 11 and 12 from Lmna∆150 (exon11∆150 and the coding
sequence of exon 12), followed by a bovine growth hormone polyadenylation transcriptional stop
signal (BGH-polyA), and an 877-bp right homology arm (part of Lmna intron 10, exon 11, intron
11 and part of exon 12).
Oocyte microinjection and implantation into pseudo-pregnant females
Hormonal superovulation was induced in 10 immature female mice (3-5 weeks old, C57BL/6J
genetic background) by intraperitoneal hormone injection. Mice first received an injection of 0.1
mL (5 IU) of pregnant mare serum gonadotropin, followed 48 h later by an injection of 0.1 mL (5
IU) of human chorionic gonadotropin. Immediately after the second injection, animals were mated
with appropriate stud males. One day after mating, females were checked for vaginal plugs, and
those with a positive result were sacrificed. Oviducts from sacrificed females were extracted and
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transferred to M2 culture medium (M7167; Sigma-Aldrich, St Louis, MO USA) containing 350
µg/mL hyaluronidase (H3884, Sigma-Aldrich). Each ampulla was localized and opened to release
the cumulus mass, and oocytes were separated after incubation at 37 ºC for 1-2 minutes (min) and
then transferred to fresh M2 medium for washing. Zygotes were incubated in Evolve-KSOM
culture medium (ZEKS-050; Zenith Biotech, Cork, Ireland) at 37 ºC in a 5% CO2/5% O2
atmosphere, until they were ready for pronuclear microinjection16. Zygotes were microinjected
with 1-2 pL of a microinjection solution containing guide RNAs, dsDNA donor template and Cas9
endonuclease (Table I in the Supplement). Zygotes were incubated overnight (o/n) at 37 ºC and
5% CO2/5% O2 in Evolve-KSOM medium (ZEKS-050; Zenith Biotech) to reach the 2-cell stage16.
Embryos at the 2-cell stage were transferred to pseudo-pregnant female mice by passing a sterile
glass needle through the infundibulum. Three weeks later, 34 pups were weaned from their
gestational mothers.
Identification of founder HGPSrev mice
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To identify mice carrying the LmnaHGPSrev allele, we extracted genomic DNA from the tails of the
34 mouse pups following a standard protocol with Proteinase K (EO0492; Thermo Fisher,
Waltham, MA USA) and performed PCR with specific primers (Table II in the Supplement Founders: PCR-1). Additional PCR reactions were run to identify mice carrying a single copy of
the mutant allele at the proper location in the Lmna locus (Table II in the Supplement Founders: PCR-2 and PCR-3). Genomic DNA from the 4 pups carrying a single copy of the
edited allele was amplified by PCR and sequenced at the Sequencing Service of the Centro
Nacional de Investigaciones Oncológicas (CNIO, Madrid, Spain) (Figure IB in the Supplement).
Genotyping of HGPSrev mice
To genotype HGPSrev, HGPSrev-Ubc-CreERT2 and HGPSrev-SM22α-Cre mice, genomic DNA
was extracted from the tail following a standard Proteinase K protocol (EO0492; Thermo Fisher)
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and PCR reactions were performed with specific primers (Table II in the Supplement Genotyping).
Isolation, immortalization and transfection of mouse embryonic fibroblasts (MEFs)
Embryos isolated at embryonic day 13.5 were minced and incubated for 20 min in 2X trypsinEDTA (0.5% Trypsin, 0.53 mM EDTA•4Na) (15400-054; Invitrogen, Carlsbad, CA USA). MEFs
from each embryo were plated separately and incubated at 37 °C in complete growth medium
(DMEM supplemented with 10% heat-inactivated fetal bovine serum, 5% non-essential amino
acids, 5% penicillin/streptomycin, and 5% L-glutamine (v/v)). To generate virus to immortalize
MEFs, HEK293T cells were seeded and transfected with pCL-Puro-SV40 LT retroviral vector
(13970; Addgene, Watertown, MA USA) and pCL-ECO retroviral packaging plasmid (kindly
provided by Dr. Manuel Serrano) using Fugene 6 (E2692; Promega, Madison, WI USA).
Supernatants containing retroviral particles were harvested every 12 h over 2 days, filtered
through 0.45 µm pores, mixed with polybrene (8 µg/mL) (9268-5G; Sigma-Aldrich), and used to
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infect 2.5-5x105 MEFs in 100 mm dishes. Infected MEFs were serially passaged to select
immortalized cells by adding 2 µg/mL puromycin (P8833, Sigma-Aldrich).
Immortalized MEFs were seeded at ≈80% confluence and were transfected with 1.5 µg
pPB CAG ER-Cre-ER IRES Zeocin17 and 4.5 µg pCMV-hyPBase18 mixed with 18 µL TransITLT1 Transfection Reagent (MIR2300; GeneFlow, Staffordshire, UK) in DMEM. Zeocin-resistant
cells were selected by incubation for at least 10 days in the presence of 400 μg/mL zeocin
(R25005; Thermo Fisher).
Longevity studies
Starting at 4-8 weeks of age, mice were weighed and inspected for health and survival twice a
week. Health status was examined by a blinded veterinarian. Animals that met humane end-point
criteria were euthanized and the deaths recorded for the survival curve analysis. The ROUT test
was performed in all survival experiments, and identified two outliers: a tamoxifen-injected
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HGPSrev-Ubc-CreERT2 mouse that died at 34 months and an oil-injected mouse that died at 17
months (in both cases, administration of tamoxifen/oil started at ≈13 months). These mice were
excluded from the survival curve and statistical analysis.
Histology and immunofluorescence
Mouse tissues were fixed in 4% formaldehyde solution (prepared from paraformaldehyde) for 24
h at 4 ºC, dehydrated through an ascending series of ethanol concentrations, and finally embedded
in paraffin and cut into serial 4-μm sections.
For immunofluorescence studies, tissue cross-sections were deparaffinized, rehydrated and
washed in PBS. Antigen retrieval was performed by boiling the sections in 10 mM sodium citrate
buffer (pH 6) for 20 min, and samples were then blocked and permeabilized for 1 h at room
temperature (RT) in PBS supplemented with 0.3% Triton X-100, 5% BSA, and 5% normal goat
serum (NGS) (005-000-001, Jackson ImmunoResearch, West Grove, PA USA). Heart samples
were additionally blocked with 100 mM glycine (5001901000; Merck, Kenilworth, NJ USA).
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Primary and secondary antibodies were diluted in PBS supplemented with 0.3% Triton X-100, 5%
BSA, and 2.5% NGS. Primary antibodies included rat monoclonal anti-CD31 (1:50, DIA-310;
Dianova, Hamburg, Germany) and rabbit polyclonal anti-progerin (1:800, generated by the
Nourshargh laboratory using peptide immunogens and standard immunization procedures). After
overnight incubation at 4ºC, samples were washed and then incubated with the appropriate
fluorescently-labeled secondary antibodies for 2 h at RT (1:400 goat anti-rat Alexa Fluor 488 A11006 or 1:400 goat anti-rabbit Alexa Fluor 647 A-21245; Invitrogen) together with anti-αsmooth muscle actin-Cy3 (1:300, C6198; Sigma-Aldrich) and Hoechst nuclear stain
(bisBenzimide H 33342 trihydrochloride, B2261; Sigma Aldrich). Samples were washed and then
mounted in Fluoromount-G imaging medium (4958-02; eBioscience, San Diego, CA USA).
Images were acquired with a Leica SP5 DMI 6000B (Leica® Microsystems, Wetzlar, Germany)
or a LSM 700 Carl Zeiss (Zeiss, Oberkochen, Germany) confocal microscope.
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For staining with hematoxylin-eosin (H&E) and Masson’s trichrome, tissue cross-sections
were deparaffinized, rehydrated and washed in PBS. Stained sections were scanned with a
NanoZoomer-RS scanner (Hamamatsu, Japan), and images were exported with NDP.view2
software (Hamamatsu) and quantified with user-customized macros in Fiji software by an operator
blinded to genotype. The thickness of the subcutaneous fat layer was qualitatively scored from 1
(thinnest) to 5 (thickest) by five independent observers and mean results are presented for each
mouse. For quantitative analysis, regions containing epidermis, hypodermis, and muscle layers
were examined by a single blinded manner observer, and the mean thickness was calculated from
10 independent measurements per mouse.
mRNA isolation and reverse transcription for PCR detection of lamin A and progerin
Total RNA was extracted from powdered mouse tissue samples using TriReagent Solution
(AM9738, Thermo Fisher) and processed by alcohol precipitation. RNA was quantified in a
NanoDrop ND-1000 spectrophotometer and 1-2 µg were transcribed to cDNA using the HighDownloaded from http://ahajournals.org by on October 27, 2021
Capacity cDNA Reverse Transcription Kit (4368814; Applied Biosystems, Foster City, CA USA).
Lamin A and progerin mRNAs were detected as previously described19. cDNA samples (100 ng)
were amplified by PCR, and products were separated on a 1.5% agarose gel. Images were
acquired with the Molecular Imager® Gel DocTM XR+ System (Bio-Rad). Band intensity was
quantified using Fiji software.
Plasma biochemistry
Blood was extracted from the mandibular sinus of live mice or from the heart (by cardiac
puncture) or infrarenal abdominal aorta of euthanized animals. For biochemical analysis, plasma
was obtained from blood samples collected in Microvette EDTA tubes (Sarstedt, Newton, NC
USA) by specialized CNIC Animal Facility staff and centrifuged at 180-200 g for 15 min at 4ºC.
Biochemical variables were analyzed using a Dimension RxL Max Integrated Chemistry System
(Siemens Healthineers, Erlangen, Germany).
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Electrocardiography (ECG)
Mice were anesthetized with 1.5-2% isoflurane, and 4 ECG electrodes were inserted
subcutaneously into the limbs. ECG was recorded in the morning for approximately 2 min using a
MP36R data acquisition workstation (Biopac Systems, Goleta, CA, USA). ECG data were
exported with AcqKnowledge software (Biopac Systems) and automatically analyzed using
custom R scripts developed to: 1) remove noise and baseline fluctuations; 2) detect heart beats,
peaks and waves; 3) exclude artifacts; and 4) calculate QT intervals and T-wave steepness.
Tamoxifen administration
Tamoxifen (4-hydroxy-tamoxifen, H6278; Sigma Aldrich) was dissolved in ethanol for cell
studies or in corn oil (C8267; Sigma Aldrich) for mouse studies. The corn-oil preparation was
incubated at 55°C until the tamoxifen was fully dissolved and then passed through a 0.22 µm
filter.
Zeocin-resistant WT and HGPSrev MEFs were exposed to 25 nM tamoxifen and protein
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lysates were prepared 24, 48 and 72 h after tamoxifen administration. Negative controls were
treated with equal volume of vehicle (ethanol) for 72 h.
Mice were randomized to tamoxifen or oil groups balanced for age and sex. For proof-ofconcept studies, ≈3-month-old HGPSrev-Ubc-CreERT2 mice received daily intraperitoneal oil or
tamoxifen injections (2 mg/day/mouse) for 10 days; the mice were sacrificed one week after
finishing the treatment and tissues were extracted for western blot analysis. For the analysis of
HGPSrev-Ubc-CreERT2 mice at different disease stages, tamoxifen administration was
commenced at different ages. For longitudinal studies of survival, health status and body-weight
evolution, the effect on early disease was assessed by starting oil/tamoxifen administration at ≈6
months of age, coinciding with the beginning of growth failure, whereas late disease was assessed
by starting treatment at ≈13 months of age, when mice had developed severe symptoms and were
close to maximum survival (Table 1). For ECG and histopathological analysis, the effect on
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intermediate disease (characterized by mild cardiovascular symptoms) was assessed by starting
treatment at ≈9 months of age. All mice received daily intraperitoneal injections (1
mg/day/mouse) over 5 days and were sacrificed at 14.5 months of age, at which point 4 out of 10
oil-injected animals had died and only 1 out of 12 tamoxifen-injected animals had died.
Statistical analysis
Quantitative data are presented as the mean ± the standard error of the mean (SEM) unless
otherwise stated. Statistical tests were applied after the determination of normal distribution
(Shapiro-Wilk normality test) and equality of variances (F test). In experiments with two groups
and normal distribution, the statistical significance of differences was assessed by unpaired twotailed Student’s t-test. For non-normally distributed data in experiments with two groups, we used
the Mann-Whitney test. In experiments with more than two groups of normally distributed
populations, we applied one-way ANOVA followed by the post hoc Tukey test. For non-normally
distributed data, the non-parametric equivalent Kruskal-Wallis test was performed. When more
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than 2 groups were assessed over time without sphericity, we used mixed-effects analysis (twoway ANOVA) with the Geisser-Greenhouse correction and Sidák’s multiple comparisons.
Kaplan-Meier survival curves were compared by the log-rank (Mantel-Cox) test. Data on bodyweight evolution were analyzed by unpaired multiple t-tests with the Holm-Sidák correction.
All statistical tests were run in GraphPad Prism® 9.0.0. Differences were considered
statistically significant when p-values were below 0.05: *, p<0.05; **, p<0.01; ***, p<0.001;
****, p<0.0001.
Results
HGPSrev mice develop progeroid symptoms and allow Cre-dependent progerin suppression
and lamin A restoration
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We used the CRISPR-Cas9 strategy to generate the HGPSrev mouse model, engineered to
ubiquitously express progerin and lamin C and to lack lamin A, while allowing progerin
suppression and lamin A restoration upon Cre recombinase activation (Figure 1A, and Figure
IA,B in the Supplement). Western blot analysis in protein lysates from the tails of founder
HGPSrev mice revealed progerin and lamin C expression and undetectable lamin A (Figure IC in
the Supplement). HGPSrev mice obtained after breeding founder mice expressed progerin in all
tissues tested, as assessed by reverse transcriptase-PCR (Figure ID in the Supplement: compare
wild-type (WT) with HGPSrev), immunofluorescence (Figure 1B, and Figure II in the
Supplement: compare WT with HGPSrev), and western blot (Figure 1C). Analysis of multiple
tissues by semiquantitative PCR showed lower progerin mRNA content in HGPSrev mice than in
LmnaG609G/G609G mice, a widely used HGPS model14 (Figure ID in the Supplement: compare
HGPSrev with G609G).
We next examined progerin farnesylation in HGPSrev and LmnaG609G/G609G mice, using WT
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mice as a negative control. Proteins were immunoprecipitated from heart lysates with an antilamin A/C antibody that recognizes lamin A/C and progerin, and samples were analyzed by
western blot and liquid chromatography coupled to targeted tandem mass spectrometry (LCMS/MS) using a high-resolution precursor-reaction monitoring (PRM) method (Figure 2A and
Supplemental Methods). Compared with the initial lysates, WT heart immunoprecipitates were
enriched in lamin A/C (Figure 2B, WT (+) versus WT Initial lysate), HGPSrev heart
immunoprecipitates were enriched in progerin and lamin C and contained no detectable lamin A
(Figure 2B, HGPSrev (+) versus HGPSrev Initial lysate), and LmnaG609G/G609G heart
immunoprecipitates were enriched in progerin and lamin A/C (Figure 2B, G9609G (+) versus
G609G Initial lysate). For PRM, we monitored a peptide common to lamin A and progerin (IC,
internal control) and peptides specific for lamin A (LA) and farnesylated progerin (FP) (Figure
2C). The MS/MS spectra confirmed the presence of the farnesyl moiety in the monitored FP
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peptide (Figure 2D). MS2 extracted ion chromatograms obtained from the time-scheduled PRM
assay showed similar amounts of FP in HGPSrev and LmnaG609G/G609G hearts relative to the total
amount of A-type lamin isoforms in these mutant strains, whereas FP was undetectable in WT
hearts (Figure 2E). Consistent with this finding, LA was strongly depleted in HGPSrev and
LmnaG609G/G609G hearts relative to WT hearts (Figure 2E).
To examine potential off-target (OT) effects in HGPSrev mice, we used the online OffSpotter tool (https://cm.jefferson.edu/Off-Spotter/). Compared with the 20 mer sgRNA used for
CRISPR/Cas9-dependent editing, this analysis identified 184 mouse genomic sequences
containing 3, 4, or 5 mismatches (2, 16, and 166 sequences, respectively) (Figure IIIA in the
Supplement). We selected 3-mismatch sequences (OT-1 and OT-2) and 6 of the 4-mismatch
sequences (OT-3, OT-4, OT-5, OT-6, OT-7, and OT-8) (Figure IIIB in the Supplement), which
were amplified by PCR using as template genomic DNA of WT and HGPSrev mice (n=5 per
genotype). The PCR products had identical DNA sequence in all mice for all genomic regions
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examined (Figure IIIC in the Supplement), indicating the absence of off-target effects.
HGPSrev mice looked normal at birth and maintained a normal appearance until aged ≈5
months, when both males and females stopped gaining weight (Figure 3A). From this age,
HGPSrev mice also exhibited kyphosis (Figure 3B) and showed a significant fat loss, as assessed
from subcutaneous fat histology (Figure 3C) and in vivo magnetic resonance imaging (MRI)
(Figure 3D). At ≈8 months of age, HGPSrev mice had below-normal levels of plasma low-density
lipoprotein, but other lipids were normal (Figure IVA in the Supplement). In ≈13-month-old
HGPSrev mice, plasma triglycerides, total cholesterol, and high-density-lipoprotein levels were
reduced (Figure IVB in the Supplement), consistent with observations in other progerinexpressing mouse models20. Progerin expression was also associated with premature death in
HGPSrev mice, which had a median lifespan of 15 months versus 26 months in WT controls
(43.5% reduction) (Figure 3E).
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HGPS patients and animal models are both characterized by VSMC loss and collagen
accumulation in the artery wall, as well as electrocardiographic alterations7, 14, 20-30. In ≈8-monthold HGPSrev mice, medial VSMC content appeared normal in both the aortic arch and thoracic
aorta (Figure 4A); however, the media of the aortic arch showed increased collagen accumulation
compared with age-matched WT mice (Figure 4B). Disease progression manifested as severe
VSMC depletion in the aortic arch and thoracic aorta in ≈13-month-old HGPSrev mice (Figure
5A), which also had a significantly elevated collagen content in both the media and adventitia of
the aortic arch and in the media of the thoracic aorta (Figure 5B). Likewise, longitudinal ECG
assessment of HGPSrev mice revealed an age-dependent reduction in T-wave steepness from 8
months of age and an increase in the QT interval from 10 months (Figure 5C). These findings
demonstrate that HGPSrev mice, like HGPS patients, appear healthy at birth and progressively
develop the main features of the human disease, including cardiovascular alterations and
premature death.
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Ubiquitous progerin suppression and lamin A restoration extends lifespan when induced in
mildly symptomatic and in severely ill HGPSrev mice
Treatment of HGPS patients has been initiated at widely differing ages and disease stages6, but
how late treatment can be started and still ameliorate symptoms remains unknown. The HGPSrev
model was designed to address this key question by taking advantage of Cre recombinase
expression to remove the progerin-expressing cassette and restore lamin A expression (Figure
1A). We first performed in vitro studies in WT and HGPSrev MEFs that were stably transfected
with a vector encoding a tamoxifen-inducible Cre recombinase and a zeocin-resistance cassette
(Figure 6A). Western blot assays of zeocin-resistant cells showed complete progerin suppression
and lamin A restoration in HGPSrev MEFs 72 hours after tamoxifen administration, with no
effects in tamoxifen-treated WT MEFs (Figure 6B). To test the system in vivo, we generated
HGPSrev-Ubc-CreERT2 mice by crossing HGPSrev mice with transgenic Ubc-CreERT2-tg/+ mice,
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which ubiquitously express a tamoxifen-inducible Cre recombinase13. Compared with agematched oil (vehicle)-injected controls, ≈3-month-old HGPSrev-Ubc-CreERT2 mice injected with
tamoxifen and sacrificed 1 week later displayed progerin downregulation and lamin A expression
in all tissues tested (Figure 6C, and quantification in Figure V in the Supplement). Tamoxifen
injection in ≈13-month-old HGPSrev-Ubc-CreERT2 mice also resulted in progerin downregulation
and lamin A restoration (Figure 6D).
To assess the impact of in vivo systemic progerin suppression and lamin A restoration
starting at different stages of HGPS progression, we defined two ages for the start of tamoxifen
administration, representing early disease (≈6 months: beginning of growth failure, one of the
earliest symptoms in HGPS patients) and late disease (≈13 months: close to maximum survival)
(Figure 7A and Table 1). Treating ≈6-month-old HGPSrev-Ubc-CreERT2 mice with tamoxifen
prevented the body-weight loss observed in oil-injected controls at late disease stages (Figure 7B,
top left graph), but body weight remained below WT values in mice older than 12 months (see
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WT in Figure 3A). Despite this, progeroid mice injected with tamoxifen at ≈6 months showed an
84.5% increase in median lifespan (p<0.0001 versus oil; experiment ongoing at the time of
manuscript submission: all oil-treated controls had died by ≈15 months of age, whereas 13 out of
22 tamoxifen-treated mice were still alive and in good health at ≈27 months of age) (Figure 7B,
bottom left graph). Beginning tamoxifen administration in ≈13-month-old HGPSrev-UbcCreERT2 mice with severe symptoms resulted in progerin downregulation and lamin A expression
(Figure 6D), and increased median lifespan by 6.7% (p<0.05 versus oil-treated mice) (Figure 7B,
bottom right graph). These results demonstrate that administering tamoxifen to HGPSrev-UbcCreERT2 mice even at very advanced disease stages significantly increases lifespan, although the
benefit is much more pronounced when progerin suppression and lamin A restoration are achieved
in mildly symptomatic mice.
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We next investigated how suppressing progerin and restoring lamin A expression affects
the cardiovascular phenotype of HGPSrev-Ubc-CreERT2 mice. Tamoxifen was administered to
HGPSrev mice at ≈9 months of age (Table 1), an intermediate disease stage when the mice
already showed a clear reduction in body weight (Figure 3A) and had begun to develop
cardiovascular alterations (Figure 4B and Figure 5C). Mice were sacrificed at ≈14.5 months of
age (at which stage 4 out of 10 oil-injected animals but only 1 out of 12 tamoxifen-injected
animals had died). Longitudinal studies showed no statistically-significant differences in ECG
parameters between both groups of mice at all ages tested (Figure 7C). In contrast, histological
analysis revealed increased VSMC number and decreased medial collagen content in the aortic
arch and thoracic aorta of tamoxifen-injected HGPSrev-Ubc-CreERT2 mice relative to oil-injected
controls, although tamoxifen did not normalize these parameters to the values seen in WT mice
(Figure 7D).
Progerin suppression and lamin A restoration in vascular smooth muscle cells and
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cardiomyocytes is sufficient to prevent vascular damage and to normalize lifespan in
HGPSrev mice
To investigate whether all affected tissues should be targeted to ameliorate disease symptoms, or
whether tissue-specific therapies would be effective, we examined the effect of suppressing
progerin and restoring lamin A only in VSMCs and cardiomyocytes, major progerin targets in
HGPS patients and animal models2, 6, 7, 14, 20-28. For this analysis, we used the SM22α-Cre
transgenic line —which allows Cre-dependent recombination in VSMCs and cardiomyocytes31—
to generate HGPSrev-SM22α-Cre mice. Immunofluorescence experiments confirmed that progerin
expression was undetectable in arterial VSMCs of HGPSrev-SM22α-Cre mice and was clearly
reduced in non-endothelial cardiac cells (69±3.8% progerin-positive cells in HGPSrev mice versus
17±2.8% in HGPSrev-SM22α-Cre mice; p<0.0001 by unpaired two-tailed Student t-test), but
remained robust in endothelial cells in aorta and heart (Figure 8A) and in non-cardiovascular
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tissues (Figure II in the Supplement). Progerin elimination and lamin A restoration were also
confirmed by western blot assays in HGPSrev-SM22α-Cre aorta and heart, whereas progerin and
lamin C expression and lack of lamin A were observed in skeletal muscle, kidney, and spleen
(Figure 8B). Although HGPSrev-SM22α-Cre mice had a higher body weight than HGPSrev
controls from 12 months of age, they were significantly thinner than age-matched WT mice from
≈17 months of age (Figure 8C). Despite this, the lifespan (Figure 8D), aortic medial VSMC
density (Figure 8E), and collagen content (Figure 8F) of HGPSrev-SM22α-Cre mice were
undistinguishable from those of WT mice. These findings complement our recent studies showing
that SM22α-Cre-driven progerin expression is sufficient to promote cardiovascular alterations in
mice20, 29, 30, 32 and demonstrate that targeting progerin in VSMCs and cardiomyocytes is sufficient
to normalize life expectancy despite the reduced body weight caused by progerin expression in
other cell types.
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Discussion
We report here the generation and characterization of HGSrev mice, a new HGPS model that
features ubiquitous progerin and lamin C expression and lack of lamin A, and that allows Credependent progerin suppression and lamin A restoration in a time- and cell-type-specific manner.
HGPSrev mice progressively develop many symptoms characteristic of the human disease,
including growth failure, lipodystrophy, VSMC loss, vascular fibrosis, electrocardiographic
anomalies, and premature death. Interestingly, we found increased collagen accumulation in the
aortic media of HGPSrev mice at ages when VSMC content still does not differ from that of WT
mice. Likewise, previous studies in 8-week-old LmnaG609G/G609G mice that still exhibited normal
VSMC content revealed alterations in genes involved in aortic fibrosis32 and higher collagen III
and lysyl oxidase expression in carotid arteries33, suggesting that progerin-induced extracellular
matrix alterations precede VSMC death.
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Unlike HGPS patients and LmnaG609G/G609G and LMNA G608G transgenic mice, two widelyused HGPS models14, 26, HGPSrev mice exhibit undetectable lamin A expression; however, mice
that ubiquitously express lamin C and lack lamin A appeared normal14, 34, 35, suggesting that lamin
A absence is unlikely to provoke progeroid symptoms in HGPSrev mice. Indeed, HGPSrev mice
develop disease symptoms more slowly and die later than homozygous LMNA G608G mice36 and
LmnaG609G/G609G mice14. Our proteomic studies in HGPSrev and LmnaG609G/G609G mice revealed
similar amounts of farnesylated progerin in both models when normalized to the total amount of
A-type lamin isoforms, but HGPSrev mice exhibited lower progerin mRNA level in all tissues
tested, which may explain, at least in part, their milder phenotype.
Because HGPS is a progressive disease and children with this condition are diagnosed after
the appearance of symptoms6, it is critical to determine the reversibility of progerin-induced
damage and the optimal time window for treatment, chief questions that remain unanswered. In
progerin-expressing mice, the progeroid phenotype can be ameliorated and lifespan increased by
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CRISPR-Cas937, 38 and base-editing approaches36 to ubiquitously correct the HGPS-causing
mutation or by the delivery of antisense oligonucleotides to block pathogenic splicing of mutant
lamin A transcripts14, 39, 40; however, these treatments were administered to asymptomatic neonates
and young animals. We found an 84.5% extension in median lifespan when progerin was
suppressed and lamin A restored in mildly symptomatic ≈6-month-old HGPSrev mice. This
benefit in survival occurred despite the below-normal body weight of these animals, consistent
with previous mouse studies and clinical trials demonstrating lifespan extension without bodyweight normalization after the administration of various treatments (e.g., 3, 8, 14, 32, 36-40). Future
studies are warranted to test whether early disease stages feature irreversible adipocyte death that
causes persistent lipodystrophy. Importantly, progerin suppression and lamin A restoration also
prolonged lifespan in severely affected progeroid HGPSrev mice. Although phenotypic
amelioration was much more pronounced when progerin suppression and lamin A restoration were
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achieved in early disease stages, these results strongly suggest that it is never too late to start
treatment for HGPS. Indeed, tipifarnib has been found to prevent the late progression of existing
cardiovascular defects when administered to progeroid mice with overt disease symptoms41.
Moreover, lonafarnib improved carotid-femoral pulse wave velocity and other outcome measures
in HGPS clinical-trial participants who started treatment at advanced disease stages8 and also
prolonged survival in a trial population with an average age of enrollment of 8.4 years3.
In a background of normal expression of endogenous wild-type Lmna, Eriksson and
collaborators generated transgenic mice with doxycycline-inducible expression of progerin in skin
or osteoblasts/odontoblasts, which showed skin or bone/teeth defects upon doxycycline
administration, respectively42, 43. Remarkably, the defects in these mutant mice were normalized
by turning off progerin expression after the appearance of the phenotype. These seminal studies in
mice demonstrated that ectopic progerin expression in skin and bone does not cause irreversible
damage to these tissues in the context of normal endogenous lamin A expression. Nonetheless, it
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is important to note that HGPS patients express below-normal levels of lamin A and that progerin
is expressed in a broad range of tissues. Moreover, a major medical problem in HGPS is severe
cardiovascular disease. Thus, uncertainty remained about the ability of progerin suppression and
lamin A restoration to halt disease progression and increase lifespan when administered only in
cardiovascular cells, the major progerin targets. We found that vascular abnormalities and
premature death are both prevented in HGPSrev-SM22α-Cre mice with progerin suppression and
lamin A restoration restricted to VSMCs and cardiomyocytes. This benefit occurred despite
sustained broad progerin expression in other cell types, which was associated with significantly
reduced body weight compared with age-matched WT mice. Although our model does not
differentiate between effects in VSMCs and cardiomyocytes, we found that lifespan extension
after ubiquitous progerin suppression and lamin A restoration in symptomatic progeroid mice was
associated with reductions in both VSMC loss and collagen accumulation in the aortic media but
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did not ameliorate electrocardiographic defects. Moreover, HGPS is not known to be a
cardiomyopathy, and massive VSMC loss and accumulation of extracellular matrix components
have been observed in the arterial wall of HGPS mouse models and patients7, 14, 20, 23, 25-30. Taken
together, these findings suggest a major role of vascular disease in HGPS. Further studies using
the HGPSrev model and Cre transgenic lines specifically targeting VSMCs or cardiomyocytes will
permit to conclusively dissect out the relative contribution of these cell types to disease
progression and premature death, which may help optimize gene therapy or RNA therapy to treat
HGPS.
Acknowledgments
We thank Simon Bartlett for English editing, and Azim Surani (University of Cambridge, UK),
Steve P. Jackson (University of Cambridge, UK), Manuel Serrano (Institute for Research in
Biomedicine, Barcelona, Spain), Mariano Barbacid (CNIO, Madrid, Spain), Carlos López-Otín
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(Universidad de Oviedo, Spain) and Enrique Lara-Pezzi (CNIC, Madrid, Spain) for providing the
pPB CAG ER-Cre-ER IRES Zeocin, pCMV-hyPBase and pCL-ECO plasmids, the Ubc-CreERT2tg/+
mice, the LmnaG609G/G609G mice, and the HEK293T cells, respectively. We also thank David
Filgueiras (Hospital Clínico San Carlos; CNIC, Spain) for advice with electrocardiography, Jacob
F Bentzon (Aarhus University, DK; CNIC, Spain) for critical reading of the manuscript, Delphine
Larrieu and Josep Vicent Forment (University of Cambridge, UK) for advice on MEF
experiments, Davide Seruggia and Lluís Montoliu (CNB-CSIC, Spain) for advice on CRISPRCas9 strategy design, and the excellent support of the CNIC Animal Facility (with special thanks
to Eva Santos, Marta García, and Belén Ricote), Histology Service, Microscopy Unit (with special
thanks to Verónica Labrador), and Advanced Imaging Unit. Author contributions: AS-L and VA
conceived, designed and planned the overall study. AS-L generated the HGPSrev mouse model.
AS-L and CE-E designed and performed experiments and analyzed and interpreted results. CG-G,
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PG, MJA-M, and RR-B carried out experiments. VF and CE-E designed, analyzed and interpreted
ECG experiments. AB, LR, and SN generated, characterized, and provided the anti-progerin
antibody. EC and JV designed and performed targeted tandem mass spectrometry and interpreted
the results. VF, MRH, LdC, BD, IB and AM provided advice and scientific discussion. VA
interpreted the results and supervised the overall study. CE-E, IB, and VA wrote the manuscript.
All authors discussed the results and critically reviewed the manuscript.
Sources of Funding
This study was supported by grants to VA from the Spanish Ministerio de Ciencia e Innovación
(MCIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033 (grants SAF201679490-R, PID2019-108489RB-I00) and the Instituto de Salud Carlos III (ISCIII; grant
AC17/00067 as coordinator of TREAT-HGPS, a project in the E-Rare Joint Transnational call,
European Union Horizon 2020 Framework Programme 2017), with co-funding from Fondo
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Europeo de Desarrollo Regional (“A way to build Europe”). Additional funding: MICIN (grant
SVP-2014-068334) and Asociación Apadrina la Ciencia-Ford España-Ford Motor Company
Fund (ASL); Fundación "la Caixa” grants LCF/BQ/DR19/1170012 (CEE) and
LCF/BQ/DE14/10320024 (VF); Comunidad Autónoma de Madrid grant 2017-T1/BMD-5247
(IB); MICIN/AEI/10.13039/501100011033 grant FJCI-2017-33299 (MRH); and Wellcome Trust
grant 098291/Z/12/Z (SN). The CNIC is supported by the MICIN, the ISCIII, the Pro-CNIC
Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by
MICIN/AEI/10.13039/501100011033).
Disclosures
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The authors declare no conflict of interest. The funders had no role in the design of the study; the
collection, analysis, or interpretation of data; the writing of the manuscript; or the decision to
publish the results.
Supplemental Materials
Supplemental Methods
Supplemental Tables I – II
Supplemental Figures I – V
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Hutchinson-Gilford progeria syndrome. Nat Med. 2021;27:536-545.
40. Puttaraju M, Jackson M, Klein S, Shilo A, Bennett CF, Gordon L, Rigo F and Misteli T.
Systematic screening identifies therapeutic antisense oligonucleotides for HutchinsonGilford progeria syndrome. Nat Med. 2021;27:526-535.
41. Capell BC, Olive M, Erdos MR, Cao K, Faddah DA, Tavarez UL, Conneely KN, Qu X,
San H, Ganesh SK et al. A farnesyltransferase inhibitor prevents both the onset and late
progression of cardiovascular disease in a progeria mouse model. Proc Nat Acad Sci USA.
2008;105:15902-15907.
42. Sagelius H, Rosengardten Y, Schmidt E, Sonnabend C, Rozell B and Eriksson M.
Reversible phenotype in a mouse model of Hutchinson-Gilford progeria syndrome. J Med
Genet. 2008;45:794-801.
43. Strandgren C, Nasser HA, McKenna T, Koskela A, Tuukkanen J, Ohlsson C, Rozell B and
Eriksson M. Transgene silencing of the Hutchinson-Gilford progeria syndrome mutation
results in a reversible bone phenotype, whereas resveratrol treatment does not show overall
beneficial effects. FASEB J. 2015;29:3193-3205.
44. Sanger F, Nicklen S and Coulson AR. DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci USA. 1977;74:5463-5467.
45. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.
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46. Bonzon-Kulichenko E, Pérez-Hernández D, Nuñez E, Martínez-Acedo P, Navarro P,
Trevisán-Herraz M, Ramos MC, Sierra S, Martínez-Martínez S, Ruiz-Meana M et al. A
robust method for quantitative high-throughput analysis of proteomes by 18O labeling.
Mol Cell Proteomics. 2011;10:M110 003335.
28
10.1161/CIRCULATIONAHA.121.055313
Table 1. Age of LmnaHGPSrev-Ubc-CreERT2 mice at the time of initiation of oil or tamoxifen
administration
Disease stage (age initiation
oil/tamoxifen administration)
Age at initiation of
oil/tamoxifen
administration
(mean ± SD)
Experiments
Early disease
(≈6-month-old; symptoms emerging)
24.3 ± 2.15 weeks
Body-weight evolution and
survival
Intermediate disease
(≈9-month-old; mild symptoms)
35.9 ± 3.2 weeks
Electrocardiography and
histopathology
Late disease
(≈13-month-old; severe symptoms)
54.4 ± 1.97 weeks
Body-weight evolution and
survival
SD, standard deviation
Downloaded from http://ahajournals.org by on October 27, 2021
29
10.1161/CIRCULATIONAHA.121.055313
Figure legends
Figure 1. LmnaHGPSrev/HGPSrev (HGPSrev) mice exhibit ubiquitous progerin expression and
undetectable lamin A expression.
(A) CRISPR-Cas9 strategy for generating HGPSrev mice (see details in Methods and
Supplemental Fig. S1A). Cre activity generates a Lmna “reverted” allele that causes progerin
suppression and lamin A restoration. (B) Representative immunofluorescence images showing
progerin expression (white) and nuclei (blue) in WT and HGPSrev mice. Scale bar, 25 µm. (C)
Western blot of lamin A/C, progerin and GAPDH in 2-month-old WT and HGPSrev mice. Six
mice of each genotype were analyzed, and representative images are shown of two mice of each
genotype. The graphs show the relative amount of progerin normalized using lamin C and
GAPDH as controls. (n=3-13 WT mice; n=4-12 HGPSrev mice). Statistical analysis was
performed by two-tailed t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. A,
Downloaded from http://ahajournals.org by on October 27, 2021
adventitia; L, lumen; M, media; SKM, skeletal muscle.
Figure 2: Targeted precursor-reaction monitoring (PRM) analysis to examine progerin
farnesylation in mouse heart lysates. (A)
Workflow for the LC-MS/MS analysis of proteins extracted from mouse hearts and
immunoprecipitated with anti-lamin A/C antibodies that recognize lamin A, lamin C, and
progerin. For each genotype, each sample was the pool of 3 hearts. WT, wild-type mice; G609G,
LmnaG609G/G609G mice; HGPSrev, LmnaHGPSrev/HGPSrev mice. (B) Western blots using anti-lamin
A/C antibody to check the enrichment of lamin A, lamin C, and progerin in the
immunoprecipitated material and supernatant (+, immunoprecipitated; -, supernatant). Controls
included samples containing only beads and antibody (CT1) and only beads and protein extract
(CT2). A 10 µL aliquot of each sample was loaded onto the gel; see details in Supplemental
30
10.1161/CIRCULATIONAHA.121.055313
Methods. (C) Surrogate peptides used to detect mature lamin A and progerin: IC, internal control
peptide (present in both lamin A and progerin); LA, lamin A peptide (specific for lamin A); FP,
farnesylated progerin peptide (specific for progerin). (D) MS2 fragmentation spectrum from FP
obtained in the PRM assay. The insert shows ion ascription to the main fragment-ion series (Cterminal y-series and N-terminal b-series). (E) MS2 extracted ion chromatograms of IC, LA, and
FP peptides obtained from the time-scheduled PRM assay for the detection of lamin A and
progerin. The ion traces were obtained using fragment ion y+9 from IC, y+8 from LA, and b+8
from FP.
Figure 3. Progeroid phenotype in LmnaHGPSrev/HGPSrev (HGPSrev) mice with ubiquitous
progerin expression. (A)
Postnatal body-weight curves (n=14 WT; n=22 HGPSrev). Differences were analyzed by
Downloaded from http://ahajournals.org by on October 27, 2021
unpaired multiple t-tests and the Holm-Sidák correction. (B) Representative images of ≈8- and
≈13-month-old mice. Scale bar, 2 cm. (C) Representative images of hematoxylin & eosin-stained
skin from ≈13-month-old mice and the results of subcutaneous fat layer (SFL) score and
thickness quantification (see Methods) (n=13-14 WT; n=11-12 HGPSrev). Statistical analysis
was performed by two-tailed t-test. Scale bar, 500 µm. (D) Representative images of sagittal
whole-body cross-sections obtained by MRI (fat shown in white) and quantification of body-fat
mass and percentage fat content in ≈13-month-old mice (n=23 WT; n=10 HGPSrev). Differences
were analyzed by two-tailed t-test. (E) Kaplan-Meier survival curve (n=13 WT; n=22 HGPSrev).
Differences were analyzed by the Mantel-Cox test. ***, p<0.001; ****, p<0.0001. Data are
mean±SEM. Each symbol represents one animal.
31
10.1161/CIRCULATIONAHA.121.055313
Figure 4. Vascular smooth muscle cell (VSMC) content and collagen deposition in the
aortas of ≈8-month-old HGPSrev mice.
(A) Representative immunofluorescence of cross-sections of aortic arch (LEFT) and thoracic
aorta (RIGHT) stained with anti-smooth muscle α-actin (SMA) antibody (red) and Hoechst
33342 (blue) to visualize VSMCs and nuclei, respectively. Graphs show quantification of VSMC
content in the media as either the percentage (%) of SMA-positive area or nuclear density (n=6-8
WT; n=5-7 HGPSrev). Scale bar, 150 µm. (B) Representative images and quantification of
Masson’s trichrome staining to visualize medial and adventitial collagen content in crosssections of aortic arch (LEFT) and thoracic aorta (RIGHT) (n=11-13 WT; n=11-13 HGPSrev).
Scale bar, 50 µm. Data are mean±SEM. Each symbol represents one animal. Statistical analysis
was performed by two-tailed t-test (*, p<0.05). A, adventitia; L, lumen; M, media.
Downloaded from http://ahajournals.org by on October 27, 2021
Figure 5: Cardiovascular abnormalities in ≈13-month-old HGPSrev mice.
(A) Representative immunofluorescence images of aortic arch (LEFT) and thoracic aorta
(RIGHT). Specimens were co-stained with anti-smooth muscle α-actin (SMA) antibody (red) and
Hoechst 33342 (blue) to visualize vascular smooth muscle cells (VSMCs) and nuclei,
respectively. Graphs show quantification of VSMC content in the media as either the percentage
(%) of SMA-positive area or nuclear density (n=4-5 WT; n=7 HGPSrev). Scale bar, 150 µm.
Data are mean±SEM. Statistical analysis was performed by two-tailed t-test (**, p<0.01; ***,
p<0.001; ****, p<0.0001). (B) Representative images and quantification of Masson’s trichrome
staining to visualize medial and adventitial collagen content in aortic arch (LEFT) and thoracic
aorta (RIGHT) of ≈13-month-old mice (n=13-17 WT; n=13-14 HGPSrev). Scale bar, 50 µm.
Data are mean±SEM. Statistical analysis was performed by two-tailed t-test (***, p<0.001;
****, p<0.0001). (C) Longitudinal electrocardiography (ECG) assessment (n=12-23 WT; n=14-
32
10.1161/CIRCULATIONAHA.121.055313
19 HGPSrev). Data are median with interquartile range±minima and maxima. Differences were
analyzed by mixed-effects analysis using the Geisser-Greenhouse correction and Sidák’s
multiple comparisons test. Differences over time within each genotype: §, p<0.05; §§, p<0.01;
§§§§, p<0.0001. Differences between genotypes at each timepoint: *, p<0.05; ***, p<0.001;
****, p<0.0001. Each symbol represents one animal. A, adventitia; M, media; L, lumen.
Figure 6. In vitro and in vivo tamoxifen-induced Cre-dependent progerin suppression and
lamin A restoration.
(A) Wild-type (WT) and HGPSrev mouse embryonic fibroblasts (MEFs) were co-transfected with
plasmids to confer resistance to zeocin and express a tamoxifen-inducible Cre recombinase. (B)
Zeocin-resistant MEFs were analyzed by western blot to examine lamin A/C, progerin and
GAPDH expression. Equal volumes of ethanol or tamoxifen (25 nM final concentration) were
added to the cells as indicated. The graph shows the relative amount of progerin and lamin A in
Downloaded from http://ahajournals.org by on October 27, 2021
HGPSrev MEFs (normalized to lamin C content). (C, D) Western blot analysis of tissues of
LmnaHGPSrev/HGPSrev Ubc-CreERT2-tg/+ mice which received vehicle (oil) or tamoxifen beginning at
the age of ≈3 months (C, n=4 each group) and ≈13 months (D, n=6 each group). Mice in C were
euthanized 1 week after oil or tamoxifen administration, and mice in D when they met human
end-point criteria. Yellow arrowheads in D indicate one animal in which tamoxifen
administration did not suppress progerin or induce lamin A and that died 2 days after the end of
tamoxifen administration (see Figure 7B, bottom right). Quantification of the relative amounts
of lamin A and progerin in the blots in C is shown in Supplemental Figure S5). The graphs in D
show the relative amount of progerin and lamin A normalized to lamin C and GAPDH content.
Statistical analysis to compare genotypes was performed by two-tailed t-test. *, p<0.05; **,
p<0.01; ***, p<0.001; ****, p<0.0001.
33
10.1161/CIRCULATIONAHA.121.055313
Figure 7: Ubiquitous progerin suppression and lamin A restoration extends lifespan in both
mildly and severely symptomatic HGPSrev-Ubc-CreERT2 mice.
(A) Experimental protocol for studies with HGPSrev-Ubc-CreERT2 mice, showing the age at
which oil or tamoxifen administration started (details in Table 1). (B) Oil or tamoxifen were
administered at ≈6 months (LEFT: n=18 Oil and n=22 Tamoxifen) or ≈13 months (RIGHT: n=720 Oil and n=9-23 Tamoxifen). The graphs show the results from two independent experiments.
Differences were analyzed by unpaired multiple t-tests and the Holm-Sidák correction in bodyweight studies and by the Mantel-Cox test in Kaplan-Meier survival curves. (C) Oil or tamoxifen
were administered at ≈9 months of age, and ECG was performed at the indicated ages (n=7-10
Oil; n=11-12 Tamoxifen). Data are medians with interquartile range±minima and maxima.
Differences were analyzed by mixed-effects analysis with the Geisser-Greenhouse correction and
Sidák’s multiple comparisons test. (D) Hematoxylin & eosin (H&E) and Masson’s trichrome
staining of aortic cross-sections from mice receiving oil/tamoxifen at ≈9 months and sacrificed at
Downloaded from http://ahajournals.org by on October 27, 2021
14.5 months of age (n=5-6 Oil; n=11 Tamoxifen). A group of age-matched untreated WT mice was
included for comparison (n=7-9). Scale bar, 50 µm. Data are mean±SEM. Differences were
analyzed by one-way ANOVA and the post-hoc Tukey test. **, p<0.01; ***, p<0.001; ****,
p<0.0001. Each symbol represents one animal. A, adventitia; L, lumen; M, media; NS, nonspecific band.
Figure 8: Normal vascular phenotype and lifespan in HGPSrev-SM22α-Cre mice with
progerin suppression and lamin A restoration restricted to vascular smooth muscle cells
(VSMCs) and cardiomyocytes.
(A) Representative immunofluorescence images of thoracic aorta and hearts of ≈13-month-old
mice. Cross-sections were co-stained with antibodies against CD31 (green), smooth muscle αactin (SMA) (red) and progerin (white) and with Hoechst 33342 (blue) to visualize endothelial
34
10.1161/CIRCULATIONAHA.121.055313
cells, VSMCs, progerin, and nuclei, respectively. (B) Western blot of lamin A/C, progerin and
GAPDH in tissues of ≈13-month-old mice. (C) Body-weight curves (n=9 WT; n=13 HGPSrev;
n=11 HGPSrev-SM22α-Cre). Differences were analyzed by unpaired multiple t-tests and the
Holm-Sídák correction. Red asterisks denote differences between HGPSrev-SM22α-Cre and
HGPSrev mice. Black asterisks denote differences between HGPSrev-SM22α-Cre and WT mice.
(D) Kaplan-Meier survival curve (n=15 WT; n=15 HGPSrev; n=11 HGPSrev-SM22α-Cre).
Median lifespan was 13.73 months in HGPSrev mice, 22.4 months in HGPSrev-SM22α-Cre
mice, and 22.97 months in WT mice. Differences were analyzed with the Mantel-Cox test. (E, F)
Representative images of aortic arch stained with hematoxylin & eosin (H&E) and Masson’s
trichrome, to quantify VSMCs and fibrosis, respectively, in ≈13-month-old WT mice (n=6),
HGPSrev mice (n=5-6), and HGPSrev-SM22α-Cre mice (n=5). Differences were analyzed by
one-way ANOVA with the post-hoc Tukey test. *, p<0.05; ****, p<0.0001. Each symbol
represents one animal. Data are mean±SEM. A, adventitia; L, lumen; M, media. Scale bars, 25
Downloaded from http://ahajournals.org by on October 27, 2021
µm (except the ones in the tile scans in panel A, where they account for 200 µm).
35
DNA repair template
(2,487 bp)
Lmna ∆150
cDNA E11-12
10
RHA
11
Cas9
gRNA
Lmna ∆150
cDNA E11-12
allele
(WT)
1
2
LmnaHGPSrev allele
(HGPSrev)
1
3
4 5
6 7 8
9
10
Intron 10
11
Progerin
cDNA
LoxP
site
Transcription
stop signal
12
Lmna “reverted” allele
2
3
4 5
6 7 8
9
10
Lmna ∆150
cDNA E11-12
STOP
Lmna+
Lmna
exons
LHA: Left Homology Arm (938 bp)
RHA: Right Homology Arm (877 bp)
12
STOP
Insert (672 bp)
LHA
STOP
A
11
Cre
12
1
2
3
4 5
9
11
10
Lamin C
Lamin C
Lamin A
Progerin
B
6 7 8
WT
HGPSrev
L
L
M
Thoracic
aorta
A
A
Adv.
M
25 µm
Progerin
Heart
Hoechst
Aorta
75
37
Heart
75
37
Skeletal
muscle
75
37
WT
HGPSrev
Lamin A
Progerin
Lamin C
WT
GAPDH
Progerin/Lamin C ratio
kDa
HGPSrev
2.5
****
2.0
**
*
****
1.5
****
***
1.0
0.5
0.0
Aorta
Heart
SKM
Kidney Spleen
Liver
37
Spleen
75
37
75
37
Progerin/GAPDH ratio
Kidney
75
Liver
Downloaded from http://ahajournals.org by on October 27, 2021
C
Liver
2.0
****
1.5
**
***
*
1.0
*
***
Heart
SKM
0.5
0.0
Aorta
Kidney Spleen
Liver
12
A
WT
G609G
Initial lysate
(pool 3 hearts per genotype)
HGPSrev
Western
blot
Immunoprecipitation of lamin A, lamin C and progerin
Supernatant (-) Immunoprecipitate (+)
LC-MS/MS analysis
1) Anti-lamin A/C antibody
binding to magnetic beads
2) Incubation with lysates
3) Immunoprecipitation of
target antigen
B
Lamin A
Lamin C
75 kDa
Lamin A
Progerin
Lamin C
75 kDa
50 kDa
50 kDa
C
LA
FP
SVGGSGGGSFGDNLVTR
AAGGAGAQSSQNCΔ
Farnesyl
Lamin A
WT mouse
Downloaded from http://ahajournals.org by on October 27, 2021
IC
C-OCH3
TVLC#GTC#GQPADK
IC
TVLC#GTC#GQPADK
Peptide
Description
RT schedule
m/z
z
Amino acid sequence
IC
Internal control peptide: common sequence to
progerin and mature Lamin A
0 - 25 min
703.8238
+2
TVLC#GTC#GQPADK
LA
Mature Lamin A peptide
25 - 50 min
783.8791
+2
SVGGSGGGSFGDNLVTR
FP
Farnesylated and O-methylated progerin peptide
50 - 100 min
670.3374
+2
AAGGAGAQSSQNCΔ
C#, carbamidomethylated Cys; CΔ, farnesylated and O-methylated Cys; PRM, precursor-reaction monitoring; RT, retention time.
40
35
b+3
25
25
b8
b10
b9
b11
y+2
20
15
b+10
b+
11
10
b+12
5
00
100
200
200
300
400
400
500
600
600
m/z
700
800
800
900
HGPSrev
19.7
19.8
703.82 > 933.41
IC
50
b+8
b+9
G609G
19.7
CH3
b12
b+7
y+ 1
30
b7
1000
1000
1100
1200
1200
Relative intensity
45
200.10
50
50
b6
983.42 b+12–NH3
1000.45
55
b5
868.39 b+11–H2O
886.40
b+5
60
b4
740.33 b+10–H2O
758.34
328.16
340.23
367.17 b+6–H2O
385.18
65
438.21 b+7–H2O
456.22
b+6
70
b3
1100.45 [M+H-farnesyl-NH3-H2O]+
1118.46 [M+H-farnesyl-NH3]+
1135.48 [M+H-farnesyl]+
b2
75
75
653.30 b+9–H2O
671.31
80
WT
y1
AAGGAGAQSSQNC
566.27 b+8–H2O
584.28
85
y2
100
b+4
454.27
90
257.12
95
E
FP
b+2
143.08
100
100
Relative abundance
D
Progerin
HGPS mouse
240
30.0
783.88 > 921.48
LA
120
30.0
30.1
670.34 > 584.28
2
74.9
74.9
FP
1
25
50
75
25
50
75
Retention time / min
25
50
75
B
***
40
30
C
WT
HGPSrev
SFL
SFL
SFL score
6
10
4
8
12
16
20
WT
HGPSrev
WT
HGPSrev
****
4
2
0
WT
E
HGPSrev
8
****
6
4
2
0
25
****
400
200
0
100
80
20
15
10
5
60
****
-43.5%
40
20
0
WT
600
****
% Survival
Downloaded from http://ahajournals.org by on Octo
Age (months)
Fat content (%)
0
SFL
average
SFLaverage
thickness
(m)
thickness(µm)
20
0
D
13 months
8 months
Body fat mass (g)
Body weight (g)
A
HGPSrev
0
0
4
8
12
16
20
24
Age (months)
28
32
A
Hoechst
SMA
M
L
A
M
L
L
WT
A
M
M
HGPSrev
A
L
L
L
M
A
M
A
Thoracic aorta
L
40
20
0.003
0.002
0.001
0.000
0
B
40
20
0.004
0.003
0.002
0.001
0.000
0
HGPSrev
WT
Masson’s Trichrome
% SMA+ area (media)
Nuclei/Area (media)
0.004
Nuclei/Area (media)
60
WT
HGPSrev
L
L
L
M
L
A
M
M
M
A
A
A
Aortic arch
6
4
2
0
80
4
60
40
20
0
3
2
1
0
WT
HGPSrev
% collagen (adventitia)
8
*
Thoracic aorta
% collagen (media)
% collagen (media)
10
% collagen (adventitia)
Downloaded from http://ahajournals.org by on October 27, 2021
+
M
% SMA area (media)
Aortic arch
60
40
20
0
A
SMA
WT
Hoechst
HGPSrev
M
A
L
M
A
L
L
M
A
A M
L
Thoracic aorta
40
30
20
10
0
0.003
0.002
0.001
0.000
B
HGPSrev
****
***
0.004
30
20
10
0.003
0.002
0.001
0.000
0
WT
HGPSrev
A
A
A
M
M
M
L
L
L
L
M
A
****
40
20
0
C
80
20
***
% collagen (media)
% collagen (adventitia)
60
60
40
20
0
% collagen (adventitia)
Thoracic aorta
Aortic arch
% collagen (media)
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Masson’s Trichrome
WT
40
Nuclei/Area (media)
**
0.004
% SMA+ area (media)
***
50
Nuclei/Area (media)
% SMA+ area (media)
Aortic arch
****
15
10
5
0
10
*
***
20
****
5
50
40
30
§
25
§§
QT (ms)
T-wave steepness (mV/s)
§§§§
60
*
****
10
13
15
10
5
0
0
4
8
10
13
4
8
Age (months)
Age (months)
WT
HGPSrev
A
B
MEFs (WT & HGPSrev)
75 kDa
Lamin A
Progerin
Lamin C
37 kDa
Progerin/Lamin C ratio
Vehicle
+ zeocin
Cre-ER (zeocin resistant)
24h
72h after tamoxifen
48h
0.4
0.8
0.3
0.6
0.2
0.4
0.1
0.2
0.0
0.0
Vehicle
24
48
72
Lamin A/Lamin C ratio
pPB-CAG-ER-CreER-IRES-Zeocin
pCMV-hyPBase
(Transposase)
GAPDH
Time after tamoxifen (h)
C
Oil
Tamoxifen
Start at ≈ 3 months
Tamoxifen
Oil
NS
75 kDa
Aorta
Heart
75 kDa
Lamin A
Progerin
Lamin C
37 kDa
GAPDH
37 kDa
NS
37 kDa
37 kDa
Oil
HGPSrev-UbCreERT2
WT
HGPSrev-UbCreERT2
Tamoxifen
HGPSrev-UbCreERT2
Start at ≈ 13 months
75 kDa
Tamoxifen
Oil
75 kDa
Lamin A
Progerin
Lamin C
Liver
Liver
37 kDa
75 kDa
75 kDa
GAPDH
Heart
Heart
37 kDa
37 kDa
37 kDa
0.6
****
***
0.4
0.2
0.0
Liver
Heart
0.8
0.6
HGPSrev-UbCreERT2
***
***
0.4
0.2
0.0
Liver Heart
Oil
0.5
**
0.4
0.3
*
0.2
0.1
0.0
Liver Heart
Tamoxifen
Lamin A/GAPDH ratio
0.8
Progerin/GAPDH ratio
Progerin/Lamin C ratio
HGPSrev-UbCreERT2
Lamin A/Lamin C ratio
Downloaded from http://ahajournals.org by on October 27, 2021
D
75 kDa
Kidney
Liver
75 kDa
0.4
*
0.3
0.2
**
0.1
0.0
Liver Heart
6 months
No
symptoms
C
20
10
15
40
30
20
10
5
10
0
0
0
4
8
12
16
20
24
28
0
4
8
Age (months)
20
24
10
28
80
80
% Survival
100
****
+84.5%
*
+6.7%
40
20
0
0
12
16
20
24
28
0
4
8
Age (months)
L
Oil
M
A
L
L
0
10
M
****
** ****
13
15
Age (months)
Masson’s Trichrome
A
80
60
L
M
****
** **
40
20
0
L
M
A
M
A
L
A
L
M
A
M
L
A
2
2
Nuclei/mm (media)
2
0
28
WT
A
A
4
5
L
M
6
24
10
H&E
M
L
Tamoxifen
Aortic arch
WT
L
A
20
Masson’s Trichrome
M
A
16
15
15
Age (months)
H&E
D
12
Nuclei/mm (media)
8
Oil
4
Thoracic aorta
0
% collagen (media)
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20
60
13
Age (months)
Oil or Tamoxifen (13 months)
100
40
16
Age (months)
Oil or Tamoxifen (6 months)
60
12
T-wave steepness (mV/s)
0
% Survival
20
Oil or Tamoxifen (13 months)
Body weight (g)
30
*
Body weight (g)
Oil or Tamoxifen (6 months)
40
WT
Tamoxifen
B
HGPSrev UBC-Cre + Tamoxifen
Death
QT (ms)
HGPSrev
UBC-Cre
Late
disease
Early disease
HGPSrev UBC-Cre + Oil
5
4
3
2
1
0
L
M
****
**** **
A
% collagen (media)
A
13 months
Oil or Tamoxifen
80
60
40
20
0
M
****
**** ***
A
B
Thoracic aorta
WT
HGPSrev
HGPSrev-SM22αCre
HGPSrevHGPSrev SM22αCre
WT
kDa
75
L
M
M
A
37
GAPDH
L
A
75
M
Heart
A
Aorta
L
Lamin A
Progerin
Lamin C
Heart
37
75
Skeletal
muscle
3
L
37
2
75
HGPSrev
Kidney
1
37
200 µm
2
Spleen
75
1
3
L
37
C
Body weight (g)
3
Downloaded from http://ahajournals.org by on October 27, 2021
2
40
30
20
**
10
0
0
4
8
1
12
16
20
24
D
100
L
200 µm
1
2
3
80
60
****
40
+63.1%
20
0
0
4
L
Myocardium
Progerin
CD31
SMA
H&E
WT
Hoechst
HGPSrev
L
12
16
20
24
HGPSrev
HGPSrev-SM22αCre
HGPSrev-SM22αCre
L
A
A
L
M
M
M
4
**** ****
3
2
1
L
M
L
A
A
L
M
M
% collagen
(media)
Masson’s
Trichrome
0
A
28
Age (months)
Endocardium
WT
A
8
Nuclei/mm2
(media)
Epicardium
E
F
28
Age (months)
% Survival
HGPSrev-SM22αCre
*
50
40
30
20
10
0
**** ****
Loïc Rolas