Investigating the Role of Phosphodiesterase 4D in 2 Adrenergic Receptor Signaling in Vivo

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Investigating the Role of Phosphodiesterase 4D
in ß2 Adrenergic Receptor Signaling in Vivo

• Christine N. Chang
• Department of Human Biology
• First Reader Andrew Patterson, MD PhD
• Second Reader Jeff Wine, PhD

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Clinical significance Congestive Heart Failure

• Occurs when the heart fails to pump enough blood
  to tissues
• Affects 4.8 million in U.S.
• Cost of treating CHF in
• 2004 was 28.8 billion
• Beta blockers are a type of medication used for
  CHF

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Introduction toCardiac ß-Adrenergic receptors

• Seven transmembrane heptahelical
• receptors
• 3 bAR subtypes (b1AR, b2AR, b3AR)

____
(Turner, 2003)

• Acute b1AR and b2AR activation enhances cardiac
  contractility in response to catecholamines such
  as epinephrine
• Stimulatory G protein (Gs) coupled pathway

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ß-Adrenergic receptors
Gs
Gs
1. Stimulatory
Stimulatory
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ß-Adrenergic receptors
Gs
Gi
2. Inhibitory
Stimulatory
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Comparing the ßARs
Acute vs Chronic activation differ
?1AR ?2AR
?2AR activation protects the murine myocardium
during continuous ?1AR stimulation

• Continuous activation is cardiotoxic
• Causes detrimental ventricular remodeling

(Freedman Lefkowitz 2004 Devic et al 2001,
Patterson et al 2004)
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ß1AR
ß2AR
?
PDZ
??
Gs
PDE4D
Gs
Gi
PDE4D
Internalization
PDE4D
PDE4D
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Hypothesis Predicted Behavior
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Methods Overview

• Baseline Heart Rate and Blood Pressure
• Assessments
• Resting awake mice
• Measured using an infrared tail sensor

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Methods HR and BP
Calibrated each time!
Mice go here!
Yes! Data
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Methods HR and BP
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Methods Overview

• Baseline Heart Rate and Blood Pressure
• Assessments
• Resting awake mice
• Measured using an infrared tail sensor

• Magnetic Resonance Imaging (MRI)
• 4.7T magnet, Stanford Lucas Center
• ECG-gated, respiratory-gated
• Animals were anesthetized using isoflurane

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Methods MRI
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Methods MRI Scout Images
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Methods MRI measurements
Contracted
Relaxed
LVEDV LVESV Stroke Volume
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Methods MRI
A humble word of advice
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Results Heart rate

• From 2 to 6 months of age, ?1/PDE4D DKO mice
  exhibited significantly lower heart rates than WT
  and PDE4D KO animals
• The heart rates of PDE4D KO and WT mice were
  similar during this time period

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Left Ventricle End Diastolic Volume

• Amount of blood filling the left ventricle
• Gives us a picture of how the heart may be
  changing
• Left ventricle dilatation observed in both
  ?1/PDE4D and PDE4D KO

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Stroke Volume

• A measure of cardiac function
• Amount of blood pumped out per beat
• LVEDV LVESV
• Trends

WT slight increase PDE4D increase b1/PDE4D
increase then decrease
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Cardiac Output

• A measure of cardiac function
• Volume of blood being pumped per minute
• CO SV HR
• Trends

Significantly elevated CO in ?1/PDE4D DKO mice
compared to WT mice at 2 months of age
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Starling Curve Integrating Data
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Starling Plot
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Ejection Fraction

• Measure of the hearts efficiency and function
• The left ventricle pumps a fraction of the blood
  it contains

• __SV__
• LVEDV

• A reduced ejection fraction indicates that
  cardiomyopathy is present

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Ejection Fraction vs Stroke Volume
WT slight increase PDE4D increase b1/PDE4D
very slight increase, BUT look closely
WT little change PDE4D decrease b1/PDE4D
decrease
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Conclusions

• Disclaimer Number of mice in the study
• Left ventricle dilatation observed in both
  ?1/PDE4D and PDE4D KO
• PDE4D
• i.e. ryanodine receptor function
• Decrease in LVEDV of ?1/PDE4D mice from 8-12
• months
• Unclear why
• Possibilities Statistical aberration
• Mice dropout

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Conclusions

• Ejection Fraction vs Stroke volume Contractility
• Starling Curve
• Heart rate ?1/PDE4D
• May imply ?2ARs are not behaving like ?1ARs in
  vivo
• Regulation of heart rate is more complicated
• Preliminary data suggest that disruption of PDE4D
  is associated with cardiac remodeling

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Many Thanks!

• Andrew Patterson
• and the amazing Patterson Lab!

Jim Wong Rani Agrawal Craig Chen Nathan
Pearl Helen Hwang Tahmina Samad Meghaan
Walsh Amy Romer Elise Perry Brandon
Penn Marissa Patterson Ashley Hawlyryshyn And Tom
Finsterbach, Matt Bruss, the Conti lab
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Questions?
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Future studies

• Pressure volume loops
• Microarray