?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
MEK-ERK pathway modulation ameliorates
disease phenotypes in a mouse model
of Noonan syndrome associated
with the Raf1L613V mutation
Xue Wu,1,2 Jeremy Simpson,3,4 Jenny H. Hong,1,2 Kyoung-Han Kim,3
Nirusha K. Thavarajah,2 Peter H. Backx,3 Benjamin G. Neel,1,2 and Toshiyuki Araki2
1Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. 2Campbell Family Cancer Research Institute,
Ontario Cancer Institute and Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada.
3Department of Physiology and Medicine, Heart and Stroke/Richard Lewar Centre, University of Toronto, Toronto, Ontario, Canada.
4Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada.
Cardiac hypertrophy is a major way by which cardiomyocytes
respond to various stresses, including abnormal neurohor-
monal stimuli, hemodynamic overload, and injury. There are
2 general types of cardiac hypertrophy (1, 2): physiological,
which is associated with exercise or pregnancy, and pathologi-
cal, whose primary cause is genetic defects (termed primary
hypertrophy); and excessive afterload, resulting from condi-
tions such as hypertension or valvular stenosis (termed sec-
ondary hypertrophy). With increased cardiac stress, cardiac
hypertrophy may initially represent a compensatory response
of the myocardium. However, chronic pathological hypertrophy
predisposes to ventricular dilatation, heart failure, arrhythmia,
and/or sudden death (3, 4). Physiological hypertrophy is typi-
cally concentric, with preservation of chamber shape, absence of
inflammation or fibrosis, and normal cardiac gene expression.
In contrast, pathological hypertrophy eventually progresses to
chamber dilatation (eccentric hypertrophy), is often associated
with fibrosis, and typically leads to the reactivation of a fetal
gene expression program characterized by increased levels of
(among others) atrial natriuretic peptide (ANP), brain natri-
uretic peptide (BNP), and β–myosin heavy chain (β-MHC) (5).
Delineating the molecular pathways that distinguish physi-
ological and pathological hypertrophy, and identifying ways to
reverse the latter, are of obvious medical importance.
Primary hypertrophic cardiomyopathy (HCM), the prototypic
genetic form of pathological hypertrophy, is a leading cause of
sudden death in the young (6). The hallmark of HCM is cardiac
hypertrophy in the absence of an obvious inciting hypertrophic
stimulus (7). Mutations in genes encoding sarcomeric proteins
(e.g., β-MHC, cardiac troponin T, and myosin-binding protein
C) account for approximately 75% of primary HCM cases. Such
mutations usually alter sarcomere structure and function and
result in mechanical, biochemical, and/or bioenergetic stresses
that activate cardiomyocyte signaling pathways to mediate the
hypertrophic phenotype (8–11). Aberrant activation of hypertro-
phic signaling pathways can themselves result in hypertrophy.
For example, germline mutations in AMPK are a rare cause of
HCM (12–14). Moreover, genetic and cellular models have iden-
tified multiple signaling systems that can cause or contribute
to pathological hypertrophy, including the calcineurin-NFAT,
PI3K-Akt-mTOR, glycogen synthase kinase–3β (GSK-3β), and
JNK pathways (1, 2, 15). The detailed mechanism by which aber-
rant activation of these pathways evokes pathological hypertro-
phy remains incompletely understood.
The RAS-RAF-MEK-ERK MAPK pathway (referred to herein
as the RAS-ERK pathway) is a central signaling cascade evoked
by multiple agonists, including growth factors (e.g., Heregulin,
Authorship?note: Xue Wu and Jeremy Simpson contributed equally to this work.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2011;121(3):1009–1025. doi:10.1172/JCI44929.
Related Commentary, page 844
1010?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
IGF-I, EGF, and PDGF), cytokines (e.g., IL-6, cardiotrophin, and
leukemia inhibitory factor [LIF]), G protein–coupled receptor
(GPCR) agonists (e.g., angiotensin II [Ang II] and β-adrenergic
agonists), and physical stimuli (e.g., mechanical stretch), in car-
diomyocytes as well as other cell types (1, 2, 16). The pathway
is initiated by activation of RAS, which requires RAS–guanine
nucleotide exchange factors (RAS-GEFs) such as SOS1 and, in
most cell types, the protein-tyrosine phosphatase SHP2 (encoded
by PTPN11). RAS recruits RAF proteins (e.g., RAF1, BRAF, and
ARAF) to the cell membrane, where they are activated and sub-
sequently form complexes with MEK1/2 and ERK1/2, aided by
scaffolds such as KSR. Activated RAF proteins phosphorylate
MEK1/2, which in turn phosphorylates ERK1/2. ERKs phosphor-
ylate cytosolic substrates and also translocate to the nucleus to
stimulate diverse gene expression programs by phosphorylating
several transcription factors (16, 17).
The role of the RAS-ERK pathway in cardiac hypertrophy has
been controversial. Some data argue that excessive activity of this
pathway causes HCM, whereas other evidence suggests involve-
ment in physiological, but not pathological, hypertrophy (18, 19).
Transgenic mice with cardiac-specific expression of oncogenic
HRas (G12V) display significant cardiac hypertrophy, decreased
contractility, diastolic dysfunction associated with intersti-
tial fibrosis, induction of cardiac fetal genes, and sudden death
(20–22), all of which are consistent with HCM. In cultured car-
diomyocytes, depletion of Erk1/2 with antisense oligonucleotides
or pharmacological inhibition of Mek1/2 attenuates the hypertro-
phic response to agonist stimulation (23, 24). Mice with cardiac-
specific overexpression of dominant-negative Raf1 have no overt
phenotype, but they are resistant to the development of cardiac
hypertrophy in response to pressure overload (25), which sug-
gests that signals from Raf1 are necessary for the hypertrophic
response. On the other hand, transgenic mice expressing an acti-
vated Mek1 allele under the control of the α-MHC promoter have
concentric hypertrophy with enhanced contractile performance,
show no signs of decompensation over time, and reportedly do
not progress to pathological hypertrophy (26). A recent study even
argued against any role for ERK1/2 in cardiac hypertrophy, as
Erk1–/–Erk2+/– mice, as well as transgenic mice with cardiac-specific
expression of dual specificity phosphatase 6 (Dusp6), an ERK1/2-
specific phosphatase, showed a normal hypertrophic response to
pressure overload and exercise (27).
Over the past 10 years, germline mutations in genes encoding sev-
eral members of the RAS-ERK pathway have been identified in a set
of related, yet distinct, human developmental syndromes (28–32),
now collectively termed the RASopathies (31, 32). These disorders,
some (but not all) of which include HCM as a syndromic phenotype,
present an opportunity to clarify the role of the RAS-ERK pathway
in cardiac hypertrophy. Noonan syndrome (NS), a relatively com-
mon autosomal-dominant disorder with an occurrence of 1 in about
1,000–2,500 live births, typically presents with proportional short
stature, facial dysmorphia, and cardiovascular abnormalities. About
25%–50% of NS patients exhibit some form of myeloproliferative dis-
order (MPD), which is usually transient and resolves spontaneously;
rarely, NS patients develop the severe childhood MPD juvenile myelo-
monocytic leukemia (JMML) or other forms of leukemia (33). Muta-
tions in PTPN11 that increase SHP2 phosphatase activity account for
approximately 50% of NS cases (34); other known NS genes include
SOS1 (~10%; refs. 35, 36), RAF1 (3%–5%; refs. 37, 38), KRAS (1%–2%;
refs. 39, 40), NRAS (<1%; ref. 41), and SHOC2 (<1%; ref. 42).
Generation of inducible Raf1L613V knockin
mice. (A) Targeting strategy. Structures
of the Raf1 locus, targeting vector, mutant
allele, and location of probes for Southern
blotting are shown. (B) Correct targeting of
ES cells. Genomic DNA from WT ES cells
and PCR-positive L613Vfl/+ ES clones was
digested with XbaI (5′ and Neo probe) or
BamHI (3′ probe) and subjected to Southern
blotting with 5′, 3′, or Neo probes. Blots with
5′ and 3′ probes were run on the same gel
but were noncontiguous (white lines). (C)
Expression of Raf1L613V allele is inducible.
RNA was isolated from WT and L613Vfl/+
ES cells with or without prior transfection of
MSCV-Cre-GFP plasmid and reverse tran-
scribed into cDNA. A PCR product, obtained
by using primers within exon 11 and at
the end of exon 16 of the Raf1 cDNA, was
digested with DraIII. Note that the mutant
allele was silent until Cre was introduced,
and then was expressed efficiently.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
Although NS patients typically have valvuloseptal defects, approx-
imately 20% have HCM (43). Moreover, different NS genes are dif-
ferentially associated with HCM. Only approximately 10% of NS
patients with PTPN11 mutations (44) and approximately 20% of
those with mutations in SOS1 (35) develop HCM. By contrast, HCM
is found in approximately 95% of patients bearing RAF1 mutations
that cause increased kinase activity (37, 38). The frequency of HCM
also varies in other RASopathies. HCM is the most frequent (~80%)
cardiovascular manifestation of LEOPARD syndrome (LS), caused
by phosphatase-inactivating mutations of PTPN11 (45–48), but also
is common (~50% in each) in Costello syndrome (CS), caused by
gain-of-function mutations in HRAS (49, 50), and cardio-facio-cuta-
neous (CFC) syndrome, caused by BRAF, MEK1, or MEK2 mutations
(51–53). Whether these differences represent differential effects of
specific RAS-ERK pathway mutations, the effects of modifiers in the
outbred human population, or both remains unclear.
Mouse models have begun to address such issues and to provide
insight into the detailed pathogenesis and potential therapeutic
approaches to these disorders. For example, we previously gen-
erated a knockin mouse model of the NS-associated Ptpn11D61G
mutation that recapitulates the major features of NS, including
short stature, facial dysmorphia, mild MPD, and valvuloseptal
defects. These mice, like most PTPN11 mutant NS patients, do
not have HCM (54). Transgenic mice expressing a different NS-
associated Ptpn11 mutant, Q79R, also show valvuloseptal defects
and facial abnormalities seen in NS patients, which are prevented
by genetic ablation of Erk1/2 and prenatal pharmacological inhi-
bition of Mek, respectively (55–57). Genetic ablation of Erk1 also
prevents the development of valvuloseptal defects in mice express-
ing a highly activated Ptpn11 mutant in endocardial cells (58).
A knockin mouse model of CS caused by the HRasG12V mutation
shows HCM, but these mice also have aortic stenosis, making it
unclear whether hypertrophy is primary or secondary (59).
Here, we have generated knockin mice expressing the kinase-acti-
vating NS mutant Raf1L613V. Similar to Ptpn11 mutant mice, mice
expressing this Raf1 allele had short stature, facial dysmorphia, and
hematological abnormalities; however, they did not have valvulos-
eptal defects, but instead developed HCM. Remarkably, nearly all
L613V/+ mice show multiple NS phenotypes. (A) Short stature in L613V/+ mice. Gross appearance of 2-month-old WT and L613V/+ male mice
and growth curves of WT (n = 45) and L613V/+ (n = 45) male mice are shown. Differences were significant at all time points (P < 0.0001, 2-way
repeated-measures ANOVA; ***P < 0.0001, Bonferroni post-test). (B) L613V/+ mice have facial dysmorphia. Gross facial appearance and repre-
sentative μCT scans of skulls from WT and L613V/+ mice. Double-headed arrows indicate inner canthal distance. See Table 1 for morphometric
measurements. (C) Cytokine-independent myeloid colonies from BM of 2-month-old mice (n = 6 per genotype). ***P < 0.0001, 2-tailed Student’s
t test. (D) Splenomegaly in L613V/+ mice. Representative gross appearance and spleen weight/BW ratio in WT (n = 25) and L613V/+ (n = 25)
mice at 4 months. ***P < 0.0001, 2-tailed Student’s t test. (E) Increased total wbcs, neutrophils (NE), and monocytes (MO) in 1-year-old L613V/+
mice (n = 8 per genotype). *P < 0.05, ***P < 0.0001, 2-tailed Student’s t test.
1012? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
phenotypic abnormalities in Raf1-mutant mice were reversed by
postnatal MEK inhibitor treatment. Our results show that different
NS genes have intrinsically distinct pathological effects and demon-
strate that enhanced MEK-ERK activity is critical for causing HCM
and other RAF1-mutant NS phenotypes. Along with the companion
study on LS-associated HCM by Marin et al. (60), these findings sug-
gest a mutation-specific approach to the treatment of RASopathies.
Generation of L613V/+ mice. Expression of an activated Raf1 mutant
during development might cause embryonic lethality. Therefore,
in order to investigate the effects of the NS-associated, kinase-
activating RAF1L613V mutant, we designed an inducible knockin
Raf1L613V allele (L613Vfl; Figure 1A). The targeting vector includ-
ed a cassette containing a splice acceptor sequence, a Raf1 cDNA
fragment encoding WT exons 13–16, and a pGK-Neo (Neo) gene.
The fusion cDNA/Neo cassette was flanked by loxP sites and was
positioned upstream of exons 13–16 of the Raf1 gene itself, with an
L613V mutation introduced into exon 16 and an HSV-TK cassette
for negative selection. In the absence of Cre recombinase (Cre), Raf1
exon 12 should be spliced to the cDNA (exon 13–16), leading to the
production of WT Raf1. When Cre is present, the floxed cassette
should be excised, evoking transcription of the mutant Raf1 allele.
The targeting construct was electroporated into G4 ES cells,
and correctly targeted clones (L613Vfl/+) were identified by PCR
and confirmed by Southern blotting (Figure 1B). We also validat-
ed the desired properties of the targeted locus in L613Vfl/+ ES
cells (Figure 1C). As expected, expression of the mutant allele was
undetectable by RT-PCR in the absence of Cre, but it was induced
effectively upon introduction of a Cre expression vector (MSCV-
GFP-Cre). Mutant Raf1 protein was also expressed at levels compa-
rable to those of WT Raf1 (data not shown, but see Supplemental
Figure 4; supplemental material available online with this article;
doi:10.1172/JCI44929DS1). Chimeras were then generated by out-
bred morula aggregation, and germline transmission was obtained.
L613Vfl/+ progeny were crossed to CMV-Cre mice, which express
Cre ubiquitously, and then to WT mice, thereby generating mice
with global Raf1L613V expression (referred to herein as L613V/+
mice) on a 129S6 × C57BL/6 mixed background.
L613V/+ mice were obtained at the expected Mendelian ratio at
weaning, which indicated that on this mixed background, Raf1L613V
expression during development is compatible with embryonic via-
bility. However, similar to mice expressing NS-associated Ptpn11
mutant alleles (54), L613V/+ mice could not be obtained after
backcrossing to C57BL/6 mice for more than 3 generations. Con-
sequently, all experiments herein were performed on the 129S6 ×
C57BL/6 mixed background.
L613V/+ mice show multiple NS phenotypes. L613V/+ newborns
showed normal size at birth (data not shown). At weaning, however,
male (Figure 2A) and female (data not shown) L613V/+ mice were
significantly smaller than their WT littermates, and they remained
shorter throughout their lives. Although their overall body pro-
portions were normal, L613V/+ mice exhibited facial dysmorphia
(Figure 2B and Table 1). Consistent with their decreased body size,
the skulls of L613V/+ mice were significantly shorter than those
of WT mice. Their skull width was increased, however, resulting
in a significantly greater width/length ratio. As a result, L613V/+
mice had a “triangular” facial appearance, with a blunter snout and
widely set eyes (increased inner canthal distance). These features are
reminiscent of the facial phenotype of mice expressing NS-associ-
ated Ptpn11 mutations (54, 57, 58) and represent the mouse equiva-
lent of the facial abnormalities seen in NS patients (61).
Like mouse models of Ptpn11 mutation–associated NS (54, 58)
and many NS patients (62), L613V/+ mice had hematological
defects. There was abnormal expansion of myeloid progenitors,
and BM from L613V/+ mice yielded factor-independent myeloid
colonies (Figure 2C). L613V/+ mice also developed splenomega-
ly, which became more severe as they aged (Figure 2D). Periph-
eral blood counts were normal at 4 months of age, but by 1 year,
L613V/+ mice had developed subtle but statistically significant
leukocytosis, neutrophilia, and monocytosis (Figure 2E) with nor-
mal hematocrit and platelet counts (data not shown).
L613V/+ mice show cardiac hypertrophy with chamber dilatation.
Unlike PTPN11 alleles, which are negatively associated with
HCM in NS patients (44) and in mouse models (54, 56, 58),
RAF1 mutations that encode proteins with increased kinase
activity are strongly associated with HCM (37, 38). Remark-
ably, L613V/+ mice showed evidence of cardiac hypertrophy as
early as 2 weeks after birth, as indicated by an increased heart
weight/BW ratio (Supplemental Figure 1A). Cardiac enlarge-
ment became even more obvious in adult L613V/+ mice, with
histological analysis revealing substantial thickening of the ven-
tricular wall and septum (Figure 3A). Increased heart size can
reflect a larger number of cardiomyocytes (e.g., as a consequence
of excess proliferation during development) and/or cardiomyo-
cyte hypertrophy. Cardiomyocyte proliferation, as measured by
BrdU incorporation assays, was comparable in E16.5 L613V/+
and WT embryos (Supplemental Figure 1B). By contrast, cross-
sectional area markedly increased — by about 35% — in cardio-
myocytes from 8-week-old L613V/+ compared with WT mice
(Figure 3B), indicative of cardiac hypertrophy.
Cardiac hypertrophy can be secondary to pressure overload
caused by stenotic valves or hypertension. Notably, mice express-
ing the NS-associated Ptpn11D61G mutation have severe valvulos-
eptal abnormalities, including atrial, atrioventricular, or ven-
tricular septal defects and double-outlet RV (54). In contrast,
valvuloseptal development, as assessed by histology, appeared
normal in 1-week-old L613V/+ mice (Supplemental Figure 1C
and data not shown). Invasive hemodynamic studies established
that ventricular pressure was actually lower in L613V/+ mice
than in WT controls (see below).
To assess cardiac morphology and function, we performed
echocardiography on L613V/+ mice and littermate controls at
2 and 4 months of age. As expected, LV diastolic posterior wall
thickness (LVPWd) was increased in L613V/+ mice (Figure 3, C
and D). Although chamber size was normal in 2-month-old mice,
by 4 months, L613V/+ hearts showed an increase in LV internal
Morphometry of skulls from 2-month-old WT and L613V/+
Inner canthal distance (mm)
22.9 ± 0.1
10.4 ± 0.1
0.46 ± 0.01
6.1 ± 0.1
21.4 ± 0.3A
10.9 ± 0.1A
0.51 ± 0.01A
6.5 ± 0.1A
Morphometric measurements from μCT scans of a cohort of 2-month-old
mice (see Figure 2B for images). AP < 0.0001, 2-tailed Student’s t test.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
end-diastolic dimension (LVIDd). LV internal end-systolic dimen-
sion (LVIDs) remained within normal limits (Figure 3, C and E),
indicating preserved or enhanced function. Consistent with the
latter interpretation, stroke volume (SV), ejection fraction (EF),
fractional shortening (FS), and cardiac output (CO) were increased
in L613V/+ mice (Table 2).
Invasive hemodynamic studies confirmed and extended these con-
clusions (Figure 3F and Table 3). L613V/+ mice showed increased
dP/dtmax, consistent with enhanced contractility, but no change in
cardiac relaxation (i.e., –dP/dt). Afterload (systolic pressure) was
slightly lower in L613V/+ mice. Although this finding ruled out
hypertension as a cause of hypertrophy in L613V/+ mice, it compli-
cated comparison of dP/dtmax values. For this reason, we also com-
pared dP/dt estimated at LV pressure (LVP) of 40 mm Hg (dP/dt@
LVP40), therefore reducing or eliminating the influence of afterload
(63). Importantly, dP/dt@LVP40 was increased in L613V/+ animals
(Figure 3F), providing conclusive evidence of increased contractility.
Moreover, there was no pressure gradient across the aortic valves of
L613V/+ mice (Table 3), ruling out aortic valve stenosis as a cause of
their cardiac hypertrophy. Our finding of eccentric cardiac hyper-
trophy in the absence of pressure overload was consistent with the
conclusion that L613V/+ mice have pathological hypertrophy.
Mice and humans with pathological hypertrophy often reactivate
specific fetal genes (5, 6). There are 2 isoforms of cardiac myosin:
α-MHC (faster kinetics; encoded by MYH6) and β-MHC (slower
kinetics; encoded by MYH7). In rodents, Myh7 is expressed mainly in
L613V/+ mice show cardiac hypertrophy with chamber dilatation. (A) Representative gross appearance and H&E-stained cross-sections (origi-
nal magnification, ×4; scale bars: 2 mm) of WT (n = 25) and L613V/+ (n = 25) hearts at 8 weeks, as well as heart weight/BW ratio (HW/BW) of
4-month-old WT and L613V/+ mice. (B) Cross-sections of cardiomyocytes (original magnification, ×400; scale bars: 100 μm). Cross-sectional
area (numbers below) was measured in WGA-strained sections from 8 week-old mice (n = 5 samples per genotype, with 200 cells counted
per sample using ImageJ). (C) Representative echocardiograms of hearts from 4-month-old mice. Arrows indicate LV diastolic dimension. (D)
LVPWd at 2 and 4 months, measured by echocardiography. n = 13 (WT); 11 (L613V/+). (E) LVIDd and LVIDs of 2- and 4-month-old WT (n = 13)
and L613V/+ (n = 11) hearts. (F) Cardiac contractility of 4-month-old WT (n = 13) and L613V/+ (n = 11) hearts, as measured by invasive hemody-
namic analysis. (G) Myh6 and Myh7 gene expression in 4-month-old WT (n = 6) and L613V/+ (n = 9) hearts, assessed by quantitative real-time
PCR. (B, D, and E–G) *P < 0.05, **P < 0.005, ***P < 0.0001, 2-tailed Student’s t test.
1014? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
late fetal life, whereas Myh6 is expressed predominantly in the adult.
Reexpression of Myh7 and a shift from α-Mhc to β-Mhc is a marker
for phenotypic reprogramming and HCM (5). Indeed, Myh6 mRNA
levels decreased significantly in L613V/+ hearts, and there was a
trend toward increased Myh7 expression (P = 0.09, 1-tailed Student’s
t test; Figure 3G). Consequently, the Myh7/Myh6 ratio increased sig-
nificantly. Expression of Nppa (encoding Anp) and Nppb (encoding
Bnp), 2 other fetal genes often associated with cardiac hypertrophy
(5, 64), was unaffected in L613V/+ hearts (data not shown).
Enhanced hypertrophic response and functional decompensation in
L613V/+ hearts following pressure overload. Although L613V/+ mice
showed cardiac hypertrophy, they displayed enhanced cardiac
function without signs of heart failure for at least a year of life. To
gain further insight into the nature of the hypertrophy in L613V/+
mice, we assessed their response to biomechanical stress by trans-
verse aortic constriction (TAC). L613V/+ mice had an unusually
high acute death rate after this procedure compared with controls
(Figure 4A). Furthermore, the hearts of surviving L613V/+ mice
showed dramatic ventricular, as well as left atrial, enlargement
compared with WT mice (Figure 4B). Although WT mice had an
approximately 45% increase in heart weight/BW ratio following
TAC, L613V/+ mice had an approximately 72% increase. L613V/+
mice also developed more severe interstitial fibrosis (Figure 4C)
and perivascular fibrosis (Figure 4D and Supplemental Figure 2A)
after TAC. We excluded 2 L613V/+ mice from analysis: by 8 weeks
of TAC, these mice had sustained a large spontaneous transmural
infarct involving approximately 30% of the ventricular free wall,
and extensive fibrosis with impaired systolic and diastolic func-
tion was evident (Supplemental Figure 2B).
These morphologic and histological findings established that
L613V/+ mice have an altered response to pressure overload. Con-
sistent with this, TAC provoked increases in LVPWd in WT and
L613V/+ mice; the increase was more pronounced in the L613V/+
mice (Figure 5A). LVIDd did not change after TAC in WT or L613V/+
mice, but remained elevated in the latter (Supplemental Figure 3A).
Most importantly, several parameters of cardiac function, includ-
ing SV and FS, deteriorated in L613V/+ mice, whereas these were
unaffected in WT mice (Figure 5B). There also was a trend toward
decreased CO in L613V/+ mice subjected to TAC, although this
did not reach statistical significance because these mice increased
their heart rate sufficiently to compensate for decreased ventricular
function (Supplemental Figure 3B). In addition, cardiac contrac-
tility (measured as either dP/dtmax or dP/dt@LVP40) decreased in
L613V/+ mice, but not in WT mice (Figure 5C). Cardiac relaxation —
as assessed by –dP/dt, normalized to mean arterial pressure (i.e.,
afterload) — was reduced comparably, whereas end-diastolic pres-
sure was increased to a similar extent in WT and L613V/+ mice
(Figure 5C and Supplemental Figure 3C). Thus, while WT mice
could adapt appropriately to pressure overload, L613V/+ mice
exhibited substantial, occasionally fatal, functional decompensa-
tion with reductions in SV, FS, and dP/dtmax and dP/dt@LVP40, con-
sistent with early stages of heart failure by 8 weeks of TAC.
The Raf1L613V mutant increases Mek and Erk activation in response to
multiple stimuli. Compared with WT RAF1, Raf1L613V has increased
kinase activity in vitro and an enhanced ability to activate MEK-ERK
in transfection studies (37, 38). We assessed the effect of Raf1L613V
expressed at endogenous levels on the RAS-ERK pathway. Con-
sistent with the earlier overexpression experiments, Mek and Erk
activation (as inferred from immunoblots with activation-specific
antibodies) was enhanced in multiple cell types expressing Raf1L613V
in response to a variety of stimuli, including LIF-stimulated ES cells
(Supplemental Figure 4A) and EGF- or PDGF-stimulated mouse
embryonic fibroblasts (MEFs; Supplemental Figure 4, B and C). Of
direct relevance to the L613V/+ cardiac phenotype, Mek and Erk
activation also were higher in L613V/+ than in WT neonatal cardio-
myocytes stimulated with receptor tyrosine kinase (heregulin-β1),
cytokine receptor (IL-6), or GPCR (Ang II) agonists (Figure 6).
Recently, cardiac fibroblasts were implicated in the genesis of car-
diac hypertrophy (65, 66); notably, L613V/+ cardiac fibroblasts also
showed enhanced agonist-stimulated Mek-Erk activation (Figure 7).
Both the quantitative and the qualitative effects of the mutant Raf1
allele on Mek and Erk activation differed in cardiomyocytes versus
cardiac fibroblasts (or MEFs) and in response to different stimuli. In
some cases, mutant Raf1 affected only the magnitude of activation,
in others, solely the duration of activation, and for still others, both
magnitude and duration. Such differences might reflect distinct
feedback responses to the agonists in various cell types.
Although it was difficult to detect Erk activation in the adult
heart under basal conditions (data not shown), basal Mek activ-
ity was significantly higher in adult L613V/+ compared with WT
hearts (Figure 8A). To compare Mek and Erk activation in vivo, we
monitored the response of WT and L613V/+ mice to pressure over-
load evoked by TAC for up to 45 minutes. Mek activation remained
significantly higher in L613V/+ hearts throughout the period of
acute TAC (Figure 8A). Erk activation was significantly higher in
L613V/+ hearts after 30 minutes of TAC compared with WT hearts,
but was similar to WT at other time points (Figure 8B).
Echocardiographic parameters in L613V/+ and WT mice at 2 and
4 months of age
Heart rate (bpm)
456 ± 19 454 ± 17
38 ± 1
56 ± 1
29 ± 1
18 ± 1
485 ± 12 485 ± 22
40 ± 1
54 ± 2
28 ± 1
19 ± 1
49 ± 3A
63 ± 2B
34 ± 2A
22 ± 2B
57 ± 3C
63 ± 2A
34 ± 1C
28 ± 2C
AP < 0.005, BP < 0.05, CP < 0.0001, 2-tailed Student’s t test.
Additional hemodynamic parameters of hearts from 4-month-old
Heart rate (bpm)
Systolic pressure (mmHg)
Diastolic pressure (mmHg)
516 ± 17
121 ± 3
4.1 ± 0.7
117 ± 3
83 ± 3
–11,010 ± 332
–118 ± 4
521 ± 17
113 ± 2A
3.9 ± 0.5
109 ± 2
77 ± 2
–11,190 ± 327
–127 ± 3
Cardiac catheterizations were performed and analyzed as described in
Methods. EDP, end-diastolic pressure; MAP, mean arterial pressure.
AP < 0.05, 2-tailed Student’s t test.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
We also assayed several signaling pathways implicated in other
models of cardiac hypertrophy/HCM by immunoblotting with
appropriate phosphospecific antibodies. Activation of the MAPK
family members JNK and p38 was comparable in Ang II–stimu-
lated neonatal cardiomyocytes and EGF-stimulated cardiac fibro-
blasts (Supplemental Figure 5, A and B). Likewise, Akt, GSK-3β,
and p70S6K activation in response to the agonists tested were
unaffected by Raf1L613V expression in either cell type (Supplemen-
tal Figure 5, A and B). Importantly, in the same experiments, Mek
and Erk activation were enhanced in L613V/+ cardiomyocytes
and cardiac fibroblasts.
MEK inhibitor treatment normalizes NS phenotypes in L613V/+ mice.
The genetics of NS and other RASopathies and the ability of
Raf1L613V to selectively enhance Mek and Erk activation by mul-
tiple agonists in cardiomyocytes and cardiac fibroblasts strongly
implicate enhanced Mek-Erk activation in the pathogenesis of
NS phenotypes, including HCM. We asked whether any of these
phenotypes could be reversed if Mek-Erk activation was normal-
ized by treatment of L613V/+ mice with a MEK inhibitor. In initial
experiments, the ATP-uncompetitive inhibitor PD0325901 (67)
or empty vehicle was injected i.p. daily to WT and L613V/+ mice
(5 mg/kg BW), beginning at 4 weeks and continuing for the suc-
ceeding 6 weeks. Importantly, at the start of the treatment period,
L613V/+ mice already showed substantial growth defects, facial
dysmorphia, and cardiac hypertrophy.
Remarkably, the body length of L613V/+ mice began to catch
up with WT mice after 1 week of treatment, and by 2 weeks,
PD0325901-treated L613V/+ mice were the same length as
untreated WT mice (Figure 9A). PD0325901-treated WT mice also
increased their body length such that by the last 2 weeks of treat-
ment, they were significantly longer than control, untreated WT
mice. Notably, however, PD0325901-treated L613V/+ mice achieved
the same final body length as did treated WT mice, which argues
that increased Mek-Erk activity is the primary cause of the growth
defect in L613V/+ mice (see Discussion). Inhibitor treatment also
increased the BW of L613V/+ mice, but surprisingly, they — as
well as treated WT mice — gained substantially more BW than did
untreated WT mice (Figure 9B). Increased BW in PD0325901-treat-
Abnormal response of L613V/+ mice to pressure overload. (A) Survival curves of WT (n = 25) and L613V/+ (n = 24) mice after TAC. **P < 0.005,
log-rank test. (B) Gross appearance of hearts and heart weight/BW ratio at 8 weeks after TAC or sham surgery. Dashed outlines demonstrate
markedly enlarged left atrium in L613V/+ compared with WT mice. **P < 0.005, ***P < 0.0001, Bonferroni post-test when ANOVA was significant;
##P < 0.005, 1-tailed Student’s t test. (C) Severe interstitial fibrosis in L613V/+ hearts (PSR staining; original magnification, ×100) at 8 weeks after
TAC. Percent pixels staining positive with PSR for interstitial fibrosis was quantified using ImageJ. n = 14 (WT); 13 (L613V/+). ***P < 0.0001,
Bonferroni post-test when ANOVA was significant; #P < 0.05, 2-tailed Student’s t test. (D) Perivascular fibrosis in hearts (PSR staining; original
magnification, ×200) at 8 weeks after TAC. Similar results were obtained when Masson Trichrome stain was used to assess fibrosis.
1016? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
ed mice was accompanied (and, presumably, in large part caused)
by an obvious increase in body fat (data not shown); thus, increased
adiposity/body mass was an unanticipated, and to our knowledge
previously unreported, side effect of PD0325901 treatment (and
possibly MEK inhibitor treatment in general; see Discussion).
PD0325901 treatment also affected the L613V/+ cardiac pheno-
type. The heart weight/BW ratio in L613V/+ mice was restored to
normal range (i.e., WT control) after treatment, whereas there was
no significant change in treated WT mice (Figure 9C). Echocardiog-
raphy (Figure 9, D and E) and invasive hemodynamic (Figure 10)
studies showed significant improvement in multiple parameters
of cardiac morphology and function. Furthermore, histological
assessment of cross-sectional area of cardiomyocytes confirmed
the normalization of cardiomyocyte size in L613V/+ mice after
treatment (Figure 9F). The significant increase in body size and
body mass caused by PD0325901 treatment potentially compli-
cates echocardiographic and invasive hemodynamic comparisons
of WT and L613V/+ mice before and after treatment. Therefore,
we compared all parameters using both nominal values and values
normalized by cube root of BW (BW1/3) (Supplemental Figure 6);
overall, the 2 analyses led to similar conclusions. First, there was a
significant reduction (toward normal) in LVPWd in L613V/+ mice
after treatment (Figure 9D); this difference was even more signifi-
cant when normalized by BW1/3 (Supplemental Figure 6A). Nomi-
nal LVIDd was unchanged in PD0325901-treated L613V/+ mice
(Figure 9E), although when this value was normalized, chamber
dilatation improved significantly, becoming comparable to that of
WT controls (Supplemental Figure 6B). Inhibitor treatment clearly
reduced the abnormal SV and FS in L613V/+ mice toward normal
levels (i.e., untreated or treated WT; Figure 10A and Supplemental
Figure 6C). There also was a strong trend toward decreased CO
in L613V/+ mice after PD0325901 treatment (Figure 10A and
Supplemental Figure 6D). Finally, the excessive cardiac contrac-
tility (dP/dt and dP/dt@LVP40) in L613V/+ mice was ameliorated
by PD0325901 treatment, whereas cardiac relaxation remained
unchanged (Figure 10B).
PD0325901 treatment did not improve the facial dysmorphia in
L613V/+ mice in the above study, most likely because skull devel-
opment had already been completed by the onset of drug adminis-
tration. We tested whether earlier, but still postnatal, MEK inhibi-
tor treatment could prevent or ameliorate L613V/+ facial defects.
Lactating female mice were injected i.p. with PD0325901 (5 mg/kg
BW) daily, beginning at P0 until weaning at P21. Weaned mice
were then injected individually with the same dose of PD0325901
Echocardiographic and hemodynamic parameters in WT and L613V/+ mice following pressure overload. (A) LVPWd at 8 weeks after TAC or
sham surgery. (B) Decreased SV and FS in L613V/+ mice after TAC. (C) Decreased cardiac contractility in L613V/+ mice after TAC. Because
LVPs were not identical in WT and L613V/+ mice (see Table 3), both dP/dtmax and dP/dt@LVP40 are shown. *P < 0.05, **P < 0.005, ***P < 0.0001,
Bonferroni post-test when ANOVA was significant; ##P < 0.005, ###P < 0.0001, 1-tailed Student’s t test. n = 12 (WT sham); 11 (L613V/+ sham);
22 (WT TAC); 13 (L613V/+ TAC).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
for another 5 weeks. As expected from our initial treatment regi-
men (Figure 9A), the growth defect in L613V/+ mice was again pre-
vented (data not shown). Remarkably, however, earlier PD0325901
treatment had a dramatic effect on the appearance of L613V/+
mice: they no longer had triangular faces, instead appearing indis-
tinguishable from treated or untreated WT controls (Figure 11A).
μCT morphometry confirmed that inner canthal distance was
reduced significantly, skull length increased, and skull width and
width/length ratio decreased in PD0325901-treated L613V/+ mice
(Figure 11B); all values were indistinguishable from those of treat-
ed or untreated WT mice by the end of the treatment period.
We describe here a knockin mouse model for NS caused by a
Raf1 gain-of-function mutation. Similar to mouse models of
Ptpn11 mutation–associated NS, Raf1L613V heterozygosity caused
proportional short stature, facial dysmorphia, and hematologi-
cal defects. Unlike phosphatase-activating Ptpn11 alleles, which
cause valvuloseptal abnormalities (54, 55, 58), L613V/+ mice
had normal valvuloseptal development and instead exhibited
eccentric cardiac hypertrophy that decompensated upon pres-
sure overload. Agonist-evoked Mek-Erk activation was enhanced
in multiple cell types without changes in several other signaling
pathways implicated in cardiac hypertrophy/HCM. Remarkably,
postnatal MEK inhibition normalized the growth, facial, and
cardiac defects in L613V/+ mice, demonstrating that continued
MEK-ERK activity is critical for causing HCM and other NS phe-
notypes and identified MEK inhibitors as potential therapeutic
agents for the treatment of NS.
RASopathies are a class of human genetic syndromes caused
by germline mutations in genes that encode components of the
RAS-ERK pathway (31, 32). Not surprisingly, these disorders share
several features, albeit with varying degrees of penetrance, yet each
also exhibits unique and characteristic phenotypes. Conceivably,
the specific mutant gene, possibly as a consequence of its position
in the pathway and susceptibility to feedback regulation, could
direct the phenotype. Alternatively, genetic modifiers in the highly
outbred human population could be determinative.
Previous mouse models suggest that both the gene and the
genetic background are important to the ultimate RASopathy phe-
notype. Clearly, different mutations in the same RASopathy gene
can result in distinguishable phenotypes: gain-of-function Ptpn11
mutations, depending on the degree of their phosphatase activity,
cause a variable spectrum of NS phenotypes (54, 55, 58). The cur-
rent study, along with a parallel analysis of knockin mice express-
ing a NS-associated Sos1E846K mutant (68), shows that mutations in
different genes that cause the same RASopathy syndrome yield dif-
ferent phenotypes: mice with phosphatase-activating Ptpn11 muta-
tions have valvuloseptal defects, but not HCM (54, 55); Sos1E846K/+
mice develop LV hypertrophy with incompletely penetrant aortic
stenosis; and L613V/+ mice exhibited HCM with normal valvulo-
Raf1L613V mutant increases Mek and Erk activation in cardiomyocytes. Cardiomyocytes prepared from neonatal WT and L613V/+ mice were
starved for 24 hours and then stimulated for the indicated number of minutes with 1 μg/ml Ang II (A), 10 ng/ml IL-6 (B), and 100 ng/ml heregulin-β1
(C). Cell lysates (15 μg protein) were immunoblotted with the indicated antibodies. Quantification of blots is also shown. 1 of 2 experiments with
comparable results is shown.
1018? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
On the other hand, mutations associated with different RASopa-
thies also have distinct effects in mice. In contrast to the NS mice
discussed above, an HRasG12V knockin mouse model of CS shows
abnormal cranial dimensions, papillomas, and angiosarcomas.
These mice have cardiac hypertrophy, but also aortic stenosis,
making it unclear whether the hypertrophy is primary or second-
ary (32, 59). As described in the companion paper by Marin et al.
(60), a mouse model of LS caused by Ptpn11Y279C indicates that,
unlike Ptpn11 alleles with increased catalytic activity, catalytically
impaired mutants develop HCM and skeletal abnormalities as well
as short stature and facial dysmorphia.
While the specific mutation plays a major role in determining
RASopathy phenotype, modifier loci also clearly contribute: just
as there is considerable phenotypic variation between family mem-
bers carrying the same NS or LS allele (69), there are differences
in disease spectrum and severity of mice with Ptpn11 (54, 58) and
Raf1 (data not shown) mutations on different strain backgrounds.
Ptpn11D61G/+ mice show incomplete penetrance of valvuloseptal
defects on mixed background and various penetrance of embry-
onic lethality on different strain backgrounds (54, 58). L613V/+
mice were obtained at the expected Mendelian ratio on mixed
background, whereas on the C57BL/6 background, this mutant
allele almost always was lethal (data not shown). All of these data
suggest that incomplete penetrance reflects strain-specific modi-
fiers. Genomic scans using SNP panels should help to determine
whether cloneable modifiers exist or whether heterosis accounts
for the variable penetrance.
The role of the RAS-ERK pathway in cardiac hypertrophy has been
controversial. Overexpression of MAPK phosphatase 1 (MKP-1)
blocks both agonist-induced hypertrophy in vitro and pressure
overload–associated hypertrophy in vivo (70). However, MKP-1
inactivates all 3 major MAPKs, so the study could not address
the specific effects of Ras-Erk pathway activation. Depletion of
ERK1/2 with antisense oligonucleotides or pharmacological inhi-
bition of MEK1/2 attenuates the hypertrophic response to ago-
nist stimulation of cultured cardiomyocytes (23, 24), consistent
with a requirement for MEK-ERK activation in the hypertrophic
response. Transgenic mice with cardiac-specific expression of
Raf1L613V mutant increases Mek and Erk activation in cardiac fibroblasts. Cardiac fibroblasts prepared from neonatal WT and L613V/+ mice were
starved for 16 hours and then stimulated for the indicated number of minutes with 50 ng/ml EGF (A), 100 ng/ml IGF-I (B), 100 ng/ml PDGF (C),
and 50 ng/ml FGF2 (D). Cell lysates (20 μg protein) were immunoblotted with the indicated antibodies. Quantification of blots is also shown.
1 of 2 experiments with comparable results is shown.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
HRasG12V display HCM associated with interstitial fibrosis and
sudden death (20–22). Cardiac-specific Nf1-deleted mice develop
marked cardiac hypertrophy, progressive cardiomyopathy, and
fibrosis as adults (71).
Conversely, other studies suggest that MEK-ERK activity is
dispensable for cardiomyocyte hypertrophy. Transgenic mice
with cardiac-restricted expression of activated Mek1 exhibit
concentric hypertrophy without signs of cardiomyopathy (26).
Although hypertrophy in this model was interpreted as physi-
ological, these mice also have impaired diastolic function and
reactivated cardiac fetal gene expression, which is more con-
sistent with pathological hypertrophy. A recent study showed
that Erk1–/–Erk2+/– mice or transgenic mice with cardiac-specific
expression of Dusp6, an Erk1/2-specific phosphatase, showed
a normal hypertrophic response to pressure overload and exer-
cise (27). In both of these lines of mice, however, residual Erk
activity cannot be excluded. Also, it is possible that Dusp6 has
other targets besides Erk1/2, which could complicate interpre-
tation of these results. Moreover, most of these earlier studies
involved cardiomyocyte-specific expression or deletion of poten-
tial hypertrophy-related genes, which excludes the potential
contribution of other cell types in the heart to the hypertrophic
response. Recent studies show that cardiac fibroblasts play key
roles in myocardial development and function (72, 73). Embry-
onic cardiac fibroblasts induce myocyte proliferation, whereas
adult cardiac fibroblasts promote myocyte hypertrophy (72) and
evoke pathological hypertrophy and fibrosis in response to dis-
ease stimuli (65, 66). Of particular note, enhanced Ras-Erk acti-
vation in cardiac fibroblasts is implicated in pathological hyper-
trophy and fibrosis caused by overexpression of the β-adrenergic
receptor in cardiomyocytes (66).
Our mouse model, in which a NS-associated Raf1 mutant was
expressed globally under normal promoter control, supports the
conclusion that excessive Ras-Erk pathway activity causes HCM. Sev-
eral lines of evidence indicated that L613V/+ mice have pathological
cardiac hypertrophy. Hypertrophy was eccentric in these mice, and
they showed the characteristic shift from Myh6 to Myh7 expression
seen in pathological hypertrophy. In response to pressure overload
by TAC, they had an unusually high death rate, presumably due to
inability to adapt to this stress or arrhythmia, while surviving mice
showed clear evidence of functional decompensation. Importantly,
in our model, unlike many previous studies (see above), the mutant
allele was expressed in both cardiomyocytes and cardiac fibroblasts,
as well as multiple other cell types. Moreover, Mek-Erk activation
was enhanced in response to multiple agonists in these cells. It will
be important to determine whether mutant expression in cardiomy-
ocytes, cardiac fibroblasts, or both is important for HCM in L613V/+
mice; our inducible Raf1 allele should facilitate such analyses. Most
importantly, postnatal MEK inhibitor treatment substantially nor-
malized the cardiac defects in L613V/+ mice, providing strong evi-
dence for the critical role of the RAS-ERK pathway in initiating and
maintaining the cardiac hypertrophic response.
Postnatal MEK inhibitor treatment also normalized the growth
defects and, if administered early enough, the facial dysmorphia in
L613V/+ mice. Notably, MEK inhibitor treatment also increased
the body length of WT mice, but there was no difference between
Enhanced Mek and Erk activation in L613V/+ hearts after pressure overload. Hearts from WT and L613V/+ mice were subjected to TAC for the
indicated number of minutes (n = 5 per group per time point), then lysed and analyzed by immunoblotting with the indicated antibodies. Erk2
levels are shown as a loading control. Each lane represents an individual animal; quantification of all samples is also shown. (A) Mek activation,
with all samples from a single time point analyzed on the same gel. (B) Representative samples of Erk activation from each time point analyzed
on the same gel. (A and B) *P < 0.05, 2-tailed Student’s t test.
1020? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
the mutant and WT treated groups in terms of final body length
after treatment. Likewise, MEK inhibitor treatment at doses that
effectively normalized L613V/+ cardiac anatomy and function had
little effect on cardiac function in WT mice. These results strongly
suggest that all of these NS phenotypes are caused by excessive
MEK-ERK activity, as opposed to the MEK inhibitor acting on a
parallel pathway to mitigate syndromic features.
Unexpectedly, we found that PD0325901 treatment caused a
significant increase in BW with an obvious increase in body fat.
Although we cannot exclude the possibility that this is an idiosyn-
cratic (i.e., off-target) effect of this specific MEK inhibitor, other evi-
dence points to a potential obesity-promoting effect of MEK-ERK
inhibition. For example, leptin activates Erk via an Shp2-dependent
pathway (74, 75), and deletion of Shp2 in postmitotic forebrain neu-
rons causes early-onset obesity with decreased ERK activation and
evidence of leptin resistance (76). We suspect that MEK inhibition
may act in analogous ways to promote obesity in our mice.
In summary, our data demonstrate a critical role of the RAS-
ERK pathway in the genesis of HCM in NS and show that NS
phenotypes can be rescued by pharmacological inhibition of
MEK inhibitor treatment rescues growth defect and cardiac hypertrophy in L613V/+ mice. Mice were injected i.p. daily with PD0325901 (PD;
5 mg/kg BW) or vehicle, starting at 4 weeks of age and for the succeeding 6 weeks. Body length (A) and BW (B) were measured weekly. Note
the rapid normalization of body length, as well as the increase in BW caused by inhibitor treatment. #P < 0.05, ##P < 0.005, ###P < 0.0001, 2-way
repeated-measures ANOVA; *P < 0.05, **P < 0.005, ***P < 0.0001, Bonferroni post-test when ANOVA was significant (black symbols, WT PD
vs. WT control; red symbols, L613V/+ PD vs. L613V/+ control). (C) Heart weight/BW ratio and (D) LVPWd were restored to within normal limits
in inhibitor-treated mice. **P < 0.005, ***P < 0.0001, Bonferroni post-test when ANOVA was significant; #P < 0.05, 1-tailed Student’s t test. (E)
LVIDd. **P < 0.005, Bonferroni post-test when ANOVA was significant; #P < 0.05, ##P < 0.005, 1-tailed Student’s t test. n = 14 (WT); 10 (L613V/+);
6 (WT PD); 14 (L613V/+ PD). (F) Cross-sectional area of cardiomyocytes (original magnification, ×400; scale bar, 100 μm), measured in WGA-
strained heart sections (n = 2 samples per group, with 200 cells counted per sample using ImageJ). ***P < 0.0001, Bonferroni post-test when
ANOVA was significant. See also Supplemental Figure 6.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
MEK1/2. Previous studies showed that genetic ablation of
Erk1/2 (55, 58) or prenatal treatment with a MEK inhibitor
(57, 68) can prevent some NS phenotypes. Although these stud-
ies provided evidence for the key role of Mek-Erk hyperactivity
in NS pathogenesis, they did not resolve whether MEK inhibi-
tion can reverse these phenotypes. Conversely, our results sug-
gest that MEK inhibition may be useful for the specific treat-
ment of Raf1 mutant NS, and possibly for other RASopathies
associated with increased MEK-ERK pathway activity. Interest-
ingly, the companion paper by Marin et al. shows that LS-asso-
ciated HCM is associated with hyperactivation of the PI3K-Akt
pathway and can be rescued by rapamycin treatment (60). Taken
together, these studies argue for a mutation-specific, personal-
ized approach to RASopathy therapy.
Generation of L613V/+ mice. To construct the targeting vector for our
inducible Raf1L613V knockin mice, a short arm containing Raf1 exon 12
(SacII-NotI genomic fragment) and a long arm including exons 13–16
(BamHI-ClaI genomic fragment) were ligated into the vector pGK Neo-
HSV-1 TK (77). The L613V (exon 16) mutation, marked by a unique
DraIII site, was introduced by site-directed mutagenesis. A splice accep-
tor sequence, a Raf1 cDNA fragment encoding wild-type exons 13–16,
and a pGK-Neo (Neo) gene were positioned after the first loxP site as
a SalI-XbaI fragment. The targeting vector was linearized with SacII
and electroporated into G4 ES cells (129S6 × C57BL/6 F1 background).
Genomic DNA, isolated from doubly G418/1-resistant and (2-deoxy-2-
fluoro-β-D-arabinofuranosyl)-5 iodouracil–resistant (FIAU-resistant)
ES clones (positive and negative selection, respectively), was screened
by PCR using primers outside and inside the targeting vector (Supple-
mental Table 1), followed by NotI digestion, which marks the targeting
vector. Homologous recombinants were confirmed by Southern blotting
using Neo and external (5′ and 3′) probes (Supplemental Table 1). For
these experiments, genomic DNA was digested with XbaI (5′ and Neo
probes) or BamHI (3′ probe).
To validate the desired properties of the targeted locus, correctly tar-
geted ES cells were transfected with a Cre-expressing plasmid (MSCV-
GFP-Cre) to excise the cDNA-Neo cassette (see below). Expression of
Raf1L613V mRNA was confirmed by RT-PCR (Supplemental Table 1), fol-
lowed by digestion with DraIII, which marks the L613V allele. Chime-
ras were generated by outbred morula aggregation (Toronto Centre of
Phenogenomics), and germline transmission was obtained (L613Vfl/+
mice). L613Vfl/+ mice (129Sv × C57BL/B6) were crossed to CMV-Cre
(C57BL/B6) mice, which express Cre ubiquitously, and then to WT
(129S6) mice to generate mice with global Raf1L613V expression (L613V/+
mice; 129Sv × C57BL/B6). Mice on a 129Sv × C57BL/B6 mixed back-
MEK inhibitor treatment normalizes cardiac function in L613V/+ mice. (A) Echocardiographic parameters of hearts after treatment with
PD0325901 as described in Figure 9. Note normalization of SV and FS, with a trend toward CO normalization. *P < 0.05, **P < 0.005,
***P < 0.0001, Bonferroni post-test when ANOVA was significant; #P < 0.05, 1-tailed Student’s t test. (B) Hemodynamic parameters, assessed by
cardiac catheterization, after PD0325901 treatment. For calculating statistical significance, significant outliers (circled data points), as assessed
by Grubbs test, were removed. *P < 0.05, Bonferroni post-test when ANOVA was significant (P = 0.09 including outliers); #P < 0.05, 1-tailed
Student’s t test (P = 0.12 including outliers). n = 14 (WT); 10 (L613V/+); 6 (WT PD); 14 (L613V/+ PD).
1022? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
ground were used for all experiments. For genotyping, genomic DNA
was prepared from tails, then subjected to PCR (Supplemental Table 1)
and digestion with DraIII.
All animal studies were approved by the University Health Network Ani-
mal Care Committee (Toronto, Ontario, Canada) and performed in accor-
dance with the standards of the Canadian Council on Animal Care.
Cell culture. ES cells were cultured on γ-irradiated MEF feeders in knock-
out DMEM (Invitrogen), containing 15% ES-tested (HyClone, Thermo Sci-
entific) FBS, 2 mM l-glutamine (Invitrogen), 0.1 mM nonessential amino
acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 100 U/ml
penicillin/streptomycin (Invitrogen), and 500 U/ml LIF (ESGRO, Chemi-
con). ES cells were transfected with MSCV-GFP-Cre plasmid using Lipo-
fectamine 2000 reagent (Invitrogen) according to the manufacturer’s
protocol. GFP-positive cells were purified by fluorescence-activated cell
sorting (FACS) at 48 hours after transfection and used for RNA isolation.
For biochemical studies, ES cells were removed
from feeders, starved in knockout DMEM con-
taining 1% FBS for 6 hours, and then stimulated
with 103 U/ml LIF before harvesting.
Primary MEFs were prepared from E13.5
embryos and cultured in DMEM (Invitrogen)
containing 10% FBS and 100 U/ml penicillin/
streptomycin (Invitrogen), as described previous-
ly (78). MEFs were starved in serum-free DMEM
for 16 hours before stimulation, and then stim-
ulated with 10 ng/ml EGF or 50 ng/ml PDGF
(both from PeproTech) before harvesting.
Neonatal mouse ventricular myocytes (neo-
natal cardiomyocytes) were isolated using
methods adapted from a previous study (79).
In brief, 1-day-old mouse hearts were harvest-
ed and predigested with 0.15 mg/ml trypsin
(Invitrogen) at 4°C for 12–16 hours, followed
by 50 U/ml type II collagenase (Worthington
Biochemical) and 0.2 mg/ml trypsin in calcium-
and bicarbonate-free Hanks buffer with HEPES
for 1–2 hours at 37°C. Noncardiomyocytes were
depleted by differential plating for 1 hour. Car-
diomyocytes were counted, seeded at 2.5 × 105
cells/ml on Falcon Primaria tissue-culture
plates (BD Biosciences), and cultured at 37°C
in DMEM/Ham’s F12 (1:1 [v/v]; Invitrogen),
10% FBS, and 100 U/ml penicillin/streptomy-
cin (Invitrogen) supplemented with 0.1 mM
bromodeoxyuridine (Sigma-Aldrich) and 20 μM
arabinosylcytosine (Sigma-Aldrich) to inhibit
rapidly proliferating cells. After 24 hours, this
medium was replaced with serum-free DMEM/
Ham’s F12 (1:1) medium supplemented with
1% insulin-transferrin-selenium supplements-X
(Invitrogen). After an additional 24 hours, car-
diomyocytes were stimulated with 10 ng/ml
IL-6 (PeproTech), 100 ng/ml heregulin-β1
(PeproTech), or 1 μg/ml Ang II (Sigma-Aldrich)
Noncardiomyocytes from the above prepara-
tion, mainly cardiac fibroblasts, were cultured
in DMEM containing 10% FBS and 100 U/ml
penicillin/streptomycin. Cardiac fibroblasts were
starved in serum-free DMEM for 16 hours, and
then stimulated with EGF (50 ng/ml), IGF-I (100 ng/ml), PDGF (100 ng/ml),
or FGF2 (100 ng/ml), all from PeproTech, before harvesting.
Body size analysis and morphometry. For growth curves, body length (anal-
nasal length) and BW were measured weekly. For skeletal morphometry,
mice were anesthetized with 2% isoflurane and scanned using a Locus Ultra
μCT scanner (GE Healthcare). 3D images of the skeleton were generated
and analyzed with GEHC MicroView software (GE Healthcare). Skull mea-
surements were made according to Jackson Laboratory standard protocols
and procedures (http://craniofacial.jax.org/standard_protocols.html).
Histology, immunohistochemistry, and BrdU incorporation assays. Hearts
for morphometry and histochemistry were fixed in the relaxed state by
infusion of 1% KCl in PBS, followed by 10% buffered formalin. Hearts
were then incubated in 10% buffered formalin overnight and embedded
in paraffin. Sections (5 μm) were prepared and stained with H&E, pic-
rosirius red (PSR), or Masson Trichrome. Cell membranes were stained
Early postnatal MEK inhibitor treatment rescues facial dysmorphia in L613V/+ mice.
Lactating female mice were injected i.p. daily with PD0325901 (5 mg/kg BW) or vehicle,
starting at P0 until weaning at P21. Weaned mice were then injected i.p. individually with
PD0325901 (5 mg/kg BW) or vehicle daily for another 5 weeks. (A) Gross facial appearance.
(B) Morphometric measurements of skulls from μCT scans. ICD, inner canthal distance.
**P < 0.005, ***P < 0.0001, Bonferroni post-test when ANOVA was significant. n = 11 (WT);
10 (L613V/+); 6 (WT PD); 7 (L613V/+ PD).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 3 March 2011
with FITC-conjugated wheat germ agglutinin (WGA; Sigma-Aldrich)
according to the manufacturer’s protocol. Nuclei were stained with
DAPI. Cross-sectional area of cardiomyocytes with centrally located
nuclei (to ensure the same plane of sectioning) was measured by using
ImageJ. 5 individual samples were analyzed per genotype, with 200 cells
measured in each.
For proliferation assays, pregnant mice (E16.5) were injected i.p. with
BrdU (100 μg/g BW) 1 hour before sacrifice. Embryos were fixed in 10%
buffered formalin overnight and embedded in paraffin. BrdU incorpora-
tion was detected using rat anti-BrdU primary antibody (1:50; Abcam).
Immune complexes were visualized using F(ab)2 biotin-conjugated donkey
anti-rat IgG (1:500; Research Diagnostics Inc.) and the Vectastain Elite ABC
Kit (Vector Laboratories). Sections were counterstained with hematoxylin.
For each sample, BrdU+ cells were counted in 10 randomly selected fields.
Hematopoietic analysis. Myeloid colony assays (in the absence of added
cytokines) were performed as described previously (80). In brief, BM
cells were suspended in MethoCult M3234 without cytokines (Stem Cell
Technologies) at 105 cells/ml, and colonies were scored after 7–9 days.
Complete blood counts were determined by using a Hemavet 850FS
Echocardiographic and hemodynamic measurements. For echocardiography
and cardiac catheterization, mice were anesthetized with isoflurane/oxy-
gen (2%:100%), and body temperature was maintained at approximately
37.5°C. Transthoracic 2D and M-mode echocardiography was performed
from the long axis view of the heart at the level of the papillary muscle
with a Visualsonics Vevo 770 imaging system (Visualsonics) equipped
with a 30-MHz linear transducer (RMV707B). Measurements of LVIDs,
LVIDd, and LVPWd were made under Time Motion-mode (TM-mode).
The papillary muscles were excluded from all measurements. Mea-
surements were averaged from at least 3 separate cardiac cycles. From
TM-mode measurements, end-diastolic volume (EDV) was calculated
as (4.5 × normalized LVIDd2); end-systolic volume (ESV) was calcu-
lated as (3.72 × normalized LDIVs2); SV was calculated as EDV — ESV;
CO was calculated as SV × heart rate; FS percentage was calculated as
(LVIDd – LVIDs)/LVIDd × 100; and EF percentage was calculated as
(EDV – ESV)/EDV × 100.
For invasive hemodynamic assessments, a 1.2F catheter (model no. FTS-
1211B-0018; Scisense Inc.) was inserted via the right carotid artery into the
LV. Hemodynamic signals were digitized at a sampling rate of 1 kHz and
recorded by computer using the MP100 imaging system and Acqknowl-
edge software (BIOPAC Systems Inc). Following recording of LV pressure,
the catheter was relocated to the ascending aortic for measurement of aor-
tic blood pressure. Mean arterial pressure was calculated as (systolic pres-
sure + diastolic pressure × 2)/3.
TAC. 8- to 9-week-old male mice (25–30 g BW) were anesthetized with 2%
isoflurane, intubated, connected to a ventilator (Harvard Apparatus), and
ventilated at a tidal volume of approximately 230 μl and 135 breaths/min. A
parasternal thoracotomy was performed to expose the transverse aorta, which
was then constricted with a 7/0 silk suture tied around a 27-gauge needle.
Pressure overload was maintained for various times as indicated in Results
and the figure legends.
MEK inhibitor treatment. N-([R]-2,3-dihydroxy-propoxy)-3,4-difluoro-2-
(2-fluoro-4-iodo-phenylamino)-benzamide (PD0325901) was synthesized
according to the disclosure in document WO2007042885(A2) (67). All
chemicals necessary for the synthesis were purchased from Sigma-Aldrich.
PD0325901 was dissolved in DMSO at a concentration of 50 mg/ml, then
resuspended in vehicle (0.5% hydroxypropyl methylcellulose with 0.2% Tween 80)
at a concentration of 0.5 mg/ml, and injected i.p. (5 mg/kg BW) daily for the
indicated times. Control mice were injected with vehicle. The same protocol
was used to inject lactating females for early postnatal treatment.
Quantitative real-time RT-PCR. Total RNA from the LV was prepared using
the RNeasy minikit (Qiagen). RNA (2 μg) was reverse transcribed using
SuperScriptIII (Invitrogen). TaqMan probe-based gene expression analy-
ses (Applied Biosystems) for Myh7, Myh6, Nppa, and Nppb (Supplemental
Table 1) were conducted according to the manufacturer’s instructions.
Each sample was measured in triplicate, and the relative expression was
normalized to GAPDH.
Biochemical analysis. Total protein extracts from cells or tissues were pre-
pared by homogenization in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 2 mM EDTA, 1% NP-40, 0.5% Na deoxycholate, and 0.1% SDS) con-
taining a protease and phosphatase inhibitor cocktail (40 μg/ml PMSF,
20 mM NaF, 1 mM Na3VO4, 10 mM β-glycerophosphate, 10 mM sodium
pyrophosphate, 2 μg/ml antipain, 2 μg/ml pepstatin A, 20 μg/ml leupeptin,
and 20 μg/ml aprotinin). Homogenates were centrifuged at 16,100 g for
15 minutes at 4°C, and the supernatants were collected. Lysates (10–25 μg
protein) were resolved by SDS-PAGE and analyzed by immunoblotting.
Antibodies for immunoblots included Raf1 (BD Biosciences); SH-PTP2
and ERK2 (C-18 and D2, respectively; Santa Cruz Biotechnology Inc.);
and phospho-MEK1/2, MEK1/2, phospho-p44/42 MAPK, phospho-S6
(Ser235/236), p38, phospho-p38, phospho-JNK1/2, Akt1, phospho-Akt
(Ser473), phospho–GSK-3β (Ser9), and phospho-P70S6K (all from Cell
Signaling Technology). Primary antibody binding was visualized by IRDye
infrared secondary antibodies using the Odyssey Infrared Imaging System
(LI-COR Biosciences). Quantification of immunoblots was performed
using Odyssey version 3.0 software.
Statistics. All data are presented as mean ± SEM. Statistical significance
was determined using 1- or 2-tailed Student’s t test, 1-way ANOVA, or 2-way
repeated measure ANOVA, as appropriate. If ANOVA was significant, indi-
vidual differences were evaluated using Bonferroni post-test. Deviation of
progeny from Mendelian frequency was assessed by χ2 test. Kaplan-Meier
survival curves were analyzed using the log-rank test. For experiments in
Figure 10B, significant outliers were identified using Grubbs test. All sta-
tistical analyses were performed with GraphPad Prism 5. For all studies, a
P value less than 0.05 was considered significant.
We thank Angel Sing (Ontario Cancer Institute) for technical
assistance. This work was supported by NIH grants HL083273
and R37CA49152 (to B.G. Neel) and Canadian Institutes of
Health Research grants 153103 (to P.H. Backx) and 106526 (to
T. Araki). This research also was funded in part by the Ontario
Ministry of Health and Long Term Care (OMOHLTC). The views
expressed do not necessarily reflect those of the OMOHLTC.
P.H. Backx is supported by Career Investigator Awards (I-6891)
of the Heart and Stroke Foundation of Ontario. X. Wu was sup-
ported by Frederick Banting and Charles Best Canada Gradu-
ate Scholarship. K.-H. Kim was supported in part by graduate
studentships from the Ontario Graduate Scholarship in Science
and Technology program.
Received for publication August 29, 2010, and accepted in revised
form December 15, 2010.
Address correspondence to: Benjamin G. Neel or Toshiyuki Araki,
Campbell Family Cancer Research Institute, Ontario Cancer
Institute and Princess Margaret Hospital, University Health Net-
work, MaRS Centre, Toronto Medical Discovery Tower, 8th Floor
Rm 8-601, 101 College Street, Toronto, Ontario, Canada M5G
1L7. Phone: 416.581.7726; Fax: 416.581.7698; E-mail: bneel@
uhnresearch.ca (B.G. Neel); firstname.lastname@example.org (T. Araki).
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