1. The
Senescence-‐Accelerated
Mouse
(SAM):
A
Murine
Model
of
Age-‐Associated
Diastolic
Dysfunc;on
Alana
L.
Reed
Advisors:
Roy
L.
Sutliff
and
Samuel
C.
Dudley
Jr.
PhD
Disserta;on
Defense
30
June
2011
2. “The
thousand
mysteries
around
us
would
not
trouble
but
interest
us,
if
only
we
had
cheerful,
healthy
hearts.”
-‐Friedrich
Wilhelm
Nietzsche
3. Aging:
demographics
and
lifespan
• The
United
States
is
experiencing
a
significant
increase
in
the
popula;on
of
older
adults
• Over
the
next
25
years,
the
number
of
Americans
over
the
age
of
65
will
double
• By
2030
there
will
be
71
million
older
adults,
comprising
20%
of
the
US
popula;on
• 80%
of
older
adults
live
with
one
or
more
chronic
medical
condi;ons
• Health
care
for
pa;ents
over
the
age
of
65
costs
approximately
five
;mes
more
than
for
a
person
under
the
age
of
65
• Healthcare
expenditures
are
projected
to
increase
by
25%
by
the
year
2030
as
a
result
of
the
growing
demographic
of
older
Americans
• Chronic
medical
condi;ons
also
decrease
the
quality
of
life
CDC,
2007
4. Theories
of
aging
• 1920’s
–
Raymond
Pearl
and
the
“rate
of
living
hypothesis”
• 1956
–
Denham
Harman’s
“free-‐radical
theory”
of
aging
• 1965
–
Hayflick
observed
senescence
in
cell
culture
What
exactly
causes
aging?
6. Mechanisms
of
aging:
telomeres
• Shortening
of
leukocyte
telomeres
correlates
with
CV
disease
(Epel
et
al.,
2009)
• Telomere
length
correlates
with
age-‐
associated
inflammatory
markers
(Blagosklonny
et
al.,
2010)
• Telomerase-‐deficient
mice
demonstrated
compromised
mitochondrial
func;on
(Sahin
et
al.,
2011)
Finkel
and
Holbrook,
2000
7. Age-‐associated
cardiovascular
changes
• Aging
is
a
major
risk
factor
for
disease
• Vascular
changes
– Dila;on
of
large
elas;c
arteries
– In;mal
media
thickening
– Increased
vascular
s;ffness
– Endothelial
dysfunc;on
• Changes
in
the
vasculature
can
set
older
individuals
up
for
heart
disease
(i.e.
hypertension)
Lakaga
and
Levy,
2003
9. Age-‐associated
cardiac
changes
LV hypertrophy
• Increased wall thickness
• Cardiomyocyte hypertrophy
• Heart failure
Diastolic dysfunction
• Decreased early diastolic
filling
• Increased late diastolic filling
• Impaired ability of LV to relax
Impaired contractility
• Decreased reserve
• Norepinephrine dysregulation
Vascular-ventricular
mismatching
• Decreased LV elastance
• Diminished cardiac reserve
Abnormal rhythmicity
• Increase in arrhythmia
• Atrial fibrillation
Vascular changes
• Dilation of large arteries
• Intimal media thickening
• Increased stiffness
• Endothelial dysfunction
10. Heart failure and diastolic dysfunction
• Half of the 5 million heart failure patients in the
US have diastolic heart failure
• Characteristics:
– Concentric remodeling
– Normal LV volume
– Slow or delayed active relaxation
– Increased passive stiffness
• Patient characteristics and risk factors:
– Elderly
– Hypertension
• Diastolic dysfunction, often clinically silent,
precedes diastolic heart failure
• Treatment strategies are limited due to a poor
understanding of the mechanism of disease,
but fibrosis is thought to play a role
11. Mechanisms
of
diastolic
dysfunc;on
• Cellular
mechanisms:
– Decline
in
SERCA2a
expression
and
ac;vity
– NCX
upregula;on
– Increased
free
ADP
– Ti;n
isoform
switching
• Extracellular
matrix:
– Collagen
deposi;on
– Changes
in
collagen
crosslinks
– Altera;on
in
MMP
and
TIMP
profiles
• Effects
external
to
LV:
– Neurohormonal
ac;va;on
– Increased
ajerload
Kass
et
al.,
2004
12. Animal
models
of
diastolic
dysfunc;on
• DOCA-‐salt
hypertension
and
pressure
overload
• Transgenic
cons;tu;vely
ac;ve
AT1
receptor
• Diabetes
and
chronic
kidney
disease
• Familial
hypertrophic
cardiomyopathy
• Advanced
age
and
senescence
13. The model: the senescence-accelerated
mouse (SAM)
• Model of spontaneous senescence that displays many
common geriatric disorders in human population
• Two series: SAMR and SAMP
• Breeders retrospectively chosen based on degree of
senescence at eight months
– Life span
– Clinical signs of aging
• Earlier onset and irreversible advancement of senescence
• SAMP have 40% shorter life span (9.7 months) than SAMR
• For our studies, we use SAMR1 and SAMP8 mice at 6
months of age
14. Cardiovascular
diseases
in
the
SAM
model
• Lipid
peroxida;on,
increased
cholesterol,
and
atherosclerosis
(Yagi,
1995
and
Fenton,
2004)
• Increased
aor;c
wall
thickness,
collagen,
and
SMC
hypertrophy
(Zhu,
2001)
• Impaired
SMC
contrac;lity,
relaxa;on,
and
endothelial
dysfunc;on
(Llorens,
2007)
• Increased
inflammatory
markers,
oxida;ve
stress,
and
endothelial
dysfunc;on
(Forman,
2010)
• Increased
mitochondrial
lipid
peroxida;on
and
increased
an;oxidant
expression
(Rodriguez,
2007)
15. Objec;ves
of
this
disserta;on
• To
inves;gate
poten;al
mechanisms
that
lead
to
the
development
of
age-‐associated
diastolic
dysfunc;on
in
a
mouse
model
of
spontaneous
accelerated
senescence
– To
establish
the
presence
of
diastolic
dysfunc;on
in
the
SAM
model
– To
evaluate
fibrosis,
and
the
role
played
by
cardiac
fibroblasts,
as
a
cause
of
diastolic
dysfunc;on
– To
examine
the
poten;al
role
played
by
oxida;ve
stress
in
age-‐associated
diastolic
dysfunc;on
and
;ssue
fibrosis
in
the
SAM
model
16. Part
I:
The
SAM
model
is
a
model
of
age-‐related
diastolic
dysfunc;on
18. SAMP8 mice show evidence of
accelerated cardiac aging
• p19 (ARF) is a tumor suppressor
protein encoded by the INK4a/
ARF locus
• p19 regulates the p53 pathway by
influencing stability of p53
– p19 inhibits MDM2, which
prevents MDM2 from targeting
p53 for degradation
• p19 plays dual roles in tumor
suppression and senescence,
since senescence requires
activation of p53
• So, p19 is a marker of
senescence and increased
expression correlates with aging
Reed
et
al.,
2011
*p<0.05
19. Heart and body weight data
SAMR1 at 6
months (n=8)
SAMP8 at 6
months (n=8)
p value
Body weight (g) 41.2 ± 1.3 42.6 ± 0.7 NS
Heart weight (mg) 110.4 ± 1.9 120.1 ± 2.2 p<0.05
HW/BW 3.6 ± 0.1 3.7 ± 0.1 NS
HW/tibial length 6.6 ± 0.1 7.0 ± 0.1 p <0.05
BW/tibial length 1.9 ± 0.04 1.9 ± 0.03 NS
Based on the heart weight/tibial length ratio, it appears there is
cardiac hypertrophy in SAMP8 mice by six months of age.
23. SAMP8 mice display evidence of diastolic
dysfunction at 6 months, but not 3 months,
of age
SAMR1
3 months old
SAMP8
3 months old
SAMR1
6 months old
SAMP8
6 months old
E/A 1.4 ± 0.03 1.4 ± 0.04 1.3 ± 0.03 1.2 ± 0.03 *§
E’ (mm/s) 28.1 ± 1.03 30.8 ± 2.0 25.7 ± 0.9 21.1 ± 0.8 §
A’ (mm/s) 20.7 ± 0.9 20.8 ± 1.7 23.3 ± 0.8 25.8 ± 1.1 §
E’/A’ 1.4 ± 0.03 1.4 ± 0.04 1.1 ± 0.02 § 0.8 ± 0.03 *§
*p<0.05 when comparison is made between SAMR1 and SAMP8 mice of the same age
§p<0.05 when comparison is made between the same type of mice at 3 and 6 months of
age
Reed
et
al.,
2011
26. What
are
the
mechanisms
driving
diastolic
dysfunc;on?
• Is
is
developing
as
a
result
of
pressure
over
load
and
hypertension?
• Is
it
driven
by
abnormal
relaxa;on
of
cardiac
myocytes?
• Are
there
abnormali;es
in
metabolism
or
other
organs
that
could
be
responsible?
• Could
cardiac
fibrosis
contribute
to
diastolic
dysfunc;on?
27. Diastolic dysfunction is unrelated to
hypertension in the SAM model
Mean arterial pressure and heart rate were measured in SAMR1
and SAMP8 mice from 3 to 6 months of age. No differences
were found, suggesting that the diastolic dysfunction observed in
this model is not secondary to hypertension.
Reed
et
al.,
2011
28. Diastolic
dysfunc;on
is
unrelated
to
cardiomyocyte
contrac;on
or
relaxa;on
Reed
et
al.,
2011
29. Metabolic profile of SAM mice
SAMR1 SAMP8 p value
(n=8) (n=8)
Bicarbonate (mM) 18.6 ± 1.6 20.4 ± 1.4 NS
Glucose (mg/dL) 251.1 ± 11.3 270.0 ± 8.4 NS
BUN (mg/dL) 15.9 ± 0.5 17.8 ± 0.4 <0.05
Creatinine (mg/dL) 0.21 ± 0.01 0.20 ± 0.0 NS
It
seems
unlikely
that
metabolic
abnormali;es
are
driving
the
development
of
diastolic
dysfunc;on
in
SAM
mice.
30. Right
heart
func;on
is
unaffected
in
SAM
mice
There
are
no
differences
between
SAMR1
and
SAMP8
mice
in
lung
weight,
RV/LV+S
ra;o,
or
RVSP,
indica;ng
that
diastolic
dysfunc;on
has
not
progressed
to
heart
failure
and
that
right
heart
func;on
has
not
been
affected.
31. Conclusions
• SAMP8
mice
undergo
accelerated
senescence
• SAMP8
mice
develop
diastolic
dysfunc;on
in
the
absence
of
systolic
dysfunc;on
by
6
months
of
age
• Diastolic
dysfunc;on
does
not
result
from
hypertension,
changes
in
cardiac
myocytes,
or
metabolic
abnormali;es
32. Part
II:
Diastolic
dysfunc;on
is
associated
with
fibrosis
in
the
SAM
model
34. Methods
• Histology
• Quan;ta;ve
real-‐;me
PCR
• Western
blot
analysis
• TGF-‐β
enzyme-‐linked
immunoassay
(ELISA)
• Cardiac
fibroblast
isola;on
and
culture
• MTT
cell
prolifera;on
assay
• Amplex®
Red
H2O2
assay
• Cardiac
fibroblast
response
to
TGF-‐β
35. Assessment of collagen: picrosirius red
staining
SAMR1 SAMP8
Using brightfield microscopy, SAMP8 mice show greater and
more intense red staining, indicating collagen accumulation at 6
months of age compared to SAMR1 controls.
36. SAMP8 mice display greater cardiac
collagen deposition
SAMP8 mice show
greater collagen
deposition in
interstitial regions
SAMP8 mice show
greater collagen
deposition in
perivascular
regions as well
SAMR1
SAMR1
SAMP8
SAMP8
Reed
et
al.,
2011
37. Increased fibrosis observed using Masson’s
trichrome staining
SAMR1
SAMR1
SAMP8
SAMP8
Reed
et
al.,
2011
*p<0.05
38. Gene expression of ECM components is
increased in SAMP8 mice
*p<0.05
• Collagen 1A1 is the major
component of scar tissue
• Collagen 3 is commonly associated
with collagen 1A1
• Fibronectin is an extracellular matrix
protein which can bind to collagen
• All three are associated with fibrosis
Reed
et
al.,
2011
39. Signaling pathways leading to fibrosis
• TGF-β is a cytokine implicated
in fibroinflammatory changes
– Fibroblast proliferation
– Extracellular matrix production
• Collagen
• Fibronectin
• TGF-β converts fibroblasts into
myofibroblasts which play a
role in organ remodeling and
fibrosis
• TGF-β can induce connective
tissue growth factor (CTGF)
– CTGF also promotes
extracellular matrix synthesis
• TGF-β and CTGF work
synergistically and are
associated with increased
collagen and fibronectin
expression
Stimuli for cytokine production
• Injury
• Pressure overload
• Neurohormonal activation
TGF-β
Cellular events
• Type I and III collagen synthesis
• Decreased proteases
• Increased TGF-b1 autoinduction
Cardiac events
• Impaired contractility
• Cardiac hypertrophy
• Dilated cardiomyopathy
• Myocardial fibrosis
Adapted
from
Lim
and
Zhu,
2006
40. Gene expression of pro-fibrotic cytokines is
increased in SAMP8 mice
*p<0.05
• TGF-β is a major pro-fibrotic cytokine that signals through the Smad
pathway
• Connective tissue growth factor (CTGF) is downstream of TGF-β and
stimulates extracellular matrix remodeling
• TGF-β and CTGF act synergistically to promote and maintain fibrosis
• Fibronectin
• Collagens 1A1 and 3A
Reed
et
al.,
2011
41. The
role
of
fibroblasts
in
fibrosis
Roles
of
the
cardiac
fibroblast
Sources
of
fibroblasts
and
myofibroblasts
Souders
et
al.,
2009
42. MTT
assay
for
fibroblast
prolifera;on
• There is no difference
in cell proliferation of
cardiac fibroblasts
from SAMR1 vs.
SAMP8 mice, so it
seems that fibrosis is
not due to increased
proliferation. n=4, p NS
43. Amplex
red
assay
for
H2O2
produc;on
• There
is
no
difference
in
hydrogen
peroxide
being
released
from
cultured
fibroblasts
from
SAMR1
vs.
SAMP8
mice.
n=4, p NS
44. Gene expression of fibrosis markers in
isolated cardiac fibroblasts
p<0.05
45. Conclusions
• SAMP8
mice
display
inters;;al
and
perivascular
cardiac
fibrosis
by
6
months
of
age
• Gene
expression
of
ECM
proteins
and
pro-‐
fibro;c
cytokines
is
increased
in
SAMP8
hearts
• Isolated
cardiac
fibroblasts
from
SAMP8
have
a
different
response
(decreased
collagen
3A)
in
response
to
TGF-‐β
s;mula;on
46. Part
III:
The
role
of
oxida;ve
stress
in
the
SAM
model
47. Oxida;ve
stress
in
SAMP
mice
• PBN
administra;on
increased
lifespan
and
prevented
protein
oxida;on
• Decreased
respiratory
control
ra;o
and
greater
metabolic
uncoupling
in
liver
and
heart
;ssue
• Increased
electron
leakage
in
brain
;ssue
• Increased
lipid
peroxida;on
in
brain
;ssue
accompanied
by
decreased
SOD
• Increased
serum
lipid
peroxide
level
and
changes
indica;ve
of
atherosclerosis
48. ROS
and
cardiac
remodeling
• MAPK
ac;va;on
leading
to
hypertrophy
• Apoptosis
• Modifica;on
of
proteins
central
to
ECC
• Ac;va;on
of
MMPs
• Sources:
– NADPH
oxidases,
XO,
mitochondria,
NOS
• An;oxidants:
– SOD,
Gaps,
catalase,
thioredoxin
Giordano,
2005
49. Oxida;ve
Stress
and
DD
• In
vitro,
increased
ROS
depresses
myocyte
contrac;lity
• Animal
models
of
CHF
have
increased
ROS
(e.g.
iron-‐
overload
cardiomyopathy)
• An;oxidants
can
improve
func;on
in
canine
model
• Mitochondrial
dysfunc;on
implicated
in
increased
ROS
Takimoto
et
al.,
2007
51. SAMP8 mice show evidence of
oxidative stress in the blood
This data suggests SAMP8 mice have increased oxidative stress in the
blood (levels were unchanged in heart tissue) compared to SAMR1
mice at 6 months of age, and this may be related to changes in Nox
proteins and/or antioxidant enzyme levels.
52. SAMP8
mice
have
increased
vascular
oxida;ve
stress
The
spin-‐probe
CMH
was
used
to
trap
O2
•-‐,
which
was
then
detected
and
quan;fied
by
ESR
in
aor;c
samples
from
6-‐month-‐old
SAMR1
and
SAMP8
mice.
SAMP8
mice
show
increased
aor;c
O2
•-‐
produc;on
compared
to
SAMR1
controls
at
6
months
of
age
(n=4,
p<0.05).
53. SAMP8
mice
show
no
difference
in
myocardial
oxida;ve
stress
O2
•-‐
was
measured
using
HPLC
analysis
with
DHE
detec;on
in
cardiac
samples
from
6-‐month-‐old
SAMR1
and
SAMP8
mice.
There
was
no
difference
in
cardiac
intracellular
O2
•-‐
between
SAMR1
and
SAMP8
mice
at
6
months
of
age
(n=8,
p=ns).
54. Do
ROS
play
a
role
in
the
SAM
model?
• Why
was
superoxide
increase
in
the
blood
and
vasculature
of
SAMP8
mice
but
not
the
heart?
• Is
superoxide
the
most
important
ROS?
• Are
an;oxidants
upregulated?
• How
might
low
levels
of
ROS
impact
signaling
pathways?
55. Nox2 and Nox4 gene expression is
increased in SAMP8 mice
However, Nox1 gene expression was unchanged.
56. Several antioxidant enzymes are
increased in SAMP8 mice
However, MnSOD, Prx3, and Sirt1 gene expression were unchanged.
57. Conclusions
• SAMP8
mice
show
increased
oxida;ve
stress
in
the
blood
and
vasculature
• Gene
expression
of
Nox2
and
Nox4
is
increased
in
the
hearts
of
SAMP8
mice
• Expression
of
catalase
and
GPX
are
also
increased
in
the
hearts
of
SAMP8
mice
• It
is
plausible
that
an;oxidants
largely
compensate
for
increased
ROS,
and
that
H2O2
may
be
the
most
important
ROS
58. Final
summary
• SAMP8 mice display diastolic dysfunction at 6
months of age
• SAMP8 mice have cardiac fibrosis, which is
thought to result in diastolic dysfunction
– Increased extracellular matrix components
– Increased pro-fibrotic cytokines
• Cardiac fibroblasts may contribute to the fibrotic
process via their response to TGF-β
• There are age-related changes in NADPH oxidase
and antioxidant gene expression, suggesting a
potential role for oxidative stress in age-associated
fibrosis and diastolic dysfunction
59. Central conclusion
The SAM model is valuable for the study of
age-related diastolic dysfunction and the
mechanisms behind the fibrotic response
that contributes to diastolic dysfunction.
60. Future
direc;ons
• Measure
TGF-‐β
receptor
expression
• Further
elucidate
the
role
of
ROS
• Examine
the
response
of
cardiac
fibroblasts
to
ROS
and
other
s;muli
• Examine
the
role
of
angiotensin
II
in
fibrosis
and
diastolic
dysfunc;on
• Inves;gate
the
role
of
immune-‐inflammatory
dysregula;on
in
promo;ng
fibrosis
• Explore
vascular
changes
in
the
SAM
model
62. Thank you!
• Sam and the Dudley lab
– Gadi Silberman, Hong Liu, Euy-Myoung Jeong, and Megan
Sturdy
• Roy and the Sutliff lab
– Erik Walp and Alex El-Ali
• Dan and the Sorescu lab
– Atsuko Tanaka and Josh Lovelock
• Committee members
– Mike Davis, Dave Harrison, and Kathy Griendling
• VA 12th floor research group
– Mike Hart, David Guidot, Tammy Murphy, Dean and Jen
Kleinhenz
• Division of Cardiology microscopy core
• BioMarkers Core Laboratory
• FRIMCORE
