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  

 

“The  thousand  mysteries  around  us  would  not  trouble  but  interest  us,  if  only  we  had  cheerful,  healthy  hearts.”    

-­‐Friedrich  Wilhelm  Nietzsche  

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  

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?  

Mechanisms  of  aging:    ROS  

Finkel  and  Holbrook,  2000  

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  

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  

Cardiovascular  aging  and  disease  

Lakaga  and  Levy,  2003  

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

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

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  

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  

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

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)  

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    

Part  I:    The  SAM  model  is  a  model  of  age-­‐related  diastolic  

dysfunc;on  

Methods  

•  Quan;ta;ve  real-­‐;me  PCR  •  Echocardiography  •  Invasive  hemodynamics  •  Func;onal  analysis  of  isolated  cardiomyocytes  •  Telemetry  

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

*p<0.05 Reed  et  al.,  2011  

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.

Measurement  of  LV  volume  and  func;on  using  echocardiography  

SAMP8 mice show no difference in cardiac structure or function at 3 or 6 months of age

SAMR1 3 months old

SAMP8 3 months old

SAMR1 6 months old

SAMP8 6 months old

LVID;s (mm) 2.9 ± 0.07 2.7 ± 0.1 2.6 ± 0.07 2.6 ± 0.07

LVID;d (mm) 4.0 ± 0.05 4.0 ± 0.1 4.0 ± 0.06 4.0 ± 0.08

LV vol;s (mm) 32.3 ± 1.8 28.1 ± 2.8 25.1 ± 1.7 24.8 ± 1.7

LV vol;d (mm) 71.6 ± 2.2 70.9 ± 4.7 69.7 ± 2.3 70.5 ± 3.6

SV (µL) 39.3 ± 1.2 42.7 ± 2.7 44.5 ± 1.0 45.7 ± 2.2

EF (%) 55.0 ± 1.6 60.5 ± 2.0 64.3 ± 1.5 65.0 ± 1.2

FS (%) 28.3 ± 1.1 32.0 ± 1.4 34.7 ± 1.1 35.3 ± 0.8

Reed  et  al.,  2011  

Doppler  echocardiography  for  the  assessment  of  diastolic  func;on  

Zile  et  al.,  2002  

SAMP8 mice display evidence of diastolic dysfunction at 6 months, but not 3 months,

of age

*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

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 *§

Reed  et  al.,  2011  

Invasive  hemodynamics:    pressure-­‐volume  loops  

Gaasch  and  Zile,  2004  

Invasive hemodynamics confirm diastolic dysfunction at 6 months of age

SAMR1 6 months old

SAMP8 6 months old

LVESP (mmHg) 85.8 ± 3.4 79.5 ± 4.0 LVEDP (mmHg) 3.4 ± 0.3 5.6 ± 0.9* dP/dtmax (mmHg/sec) 8093 ± 721 7534 ± 788 dP/dtmin (mmHg/sec) -9138 ± 832 -9089 ± 1055 Tau-Glantz (ms) 8.5 ± 0.6 8.7 ± 0.7 Tau-Weiss (ms) 5.1 ± 0.3 5.7 ± 0.4 EDPVR (mmHg/µL) 0.5 ± 0.05 0.8 ± 0.1* ESPVR (mmHg/µL) 5.9 ± 0.6 7.9 ± 1.0

*p < 0.05 compared to SAMR1 Reed  et  al.,  2011  

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?  

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  

Diastolic  dysfunc;on  is  unrelated  to  cardiomyocyte  contrac;on  or  relaxa;on  

Reed  et  al.,  2011  

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.  

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.  

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  

Part  II:    Diastolic  dysfunc;on  is  associated  with  fibrosis  in  the  

SAM  model  

Aging,  fibrosis,  and  cardiac  disease  

Chen  and  Frangogiannis,  2010  

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  

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.

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  

Increased fibrosis observed using Masson’s trichrome staining

SAMR1

SAMR1

SAMP8

SAMP8

*p<0.05 Reed  et  al.,  2011  

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  

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

Cellular events

• Type I and III collagen synthesis

• Decreased proteases

• Increased TGF-b1 autoinduction

Cardiac events

• Impaired contractility

• Cardiac hypertrophy

• Dilated cardiomyopathy

• Myocardial fibrosis

TGF-β

Adapted  from  Lim  and  Zhu,  2006  

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  

The  role  of  fibroblasts  in  fibrosis  

Roles  of  the  cardiac  fibroblast  Sources  of  fibroblasts  and  myofibroblasts  

Souders  et  al.,  2009  

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

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

Gene expression of fibrosis markers in isolated cardiac fibroblasts

p<0.05

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  

Part  III:    The  role  of  oxida;ve  stress  in  the  SAM  model  

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  

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  

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  

Methods  

•  High-­‐performance  liquid  chromatography  (HPLC)  

•  Electron  spin  resonance  (ESR)  spectroscopy  •  Quan;ta;ve  real-­‐;me  PCR  

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.

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).    

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).    

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?  

Nox2 and Nox4 gene expression is increased in SAMP8 mice

However, Nox1 gene expression was unchanged.

Several antioxidant enzymes are increased in SAMP8 mice

However, MnSOD, Prx3, and Sirt1 gene expression were unchanged.

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  

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

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.

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  

Ques;ons  and  discussion  

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

With  Much  Apprecia;on!