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Background
The identification of single gene mutations causing congenital forms of
hypertrophic and dilated cardiomyopathy has given critical insight into the
genetic events that can lead to characteristic features of heart failure in
humans. Nevertheless, there is still a large gap in our understanding of how
genetic mutations lead to a hypertrophic or dilated phenotype.
Inducing cardiac hypertrophy in vitro and in vivo
The Izumo laboratory has extensive experience in using cardiomyocyte cell
culture to study specific features of the hypertrophic response in vitro. We and
others have identified signaling pathways activated by mechanical stretch
(Sadoshima & Izumo, 1997) and different growth factor stimuli such as
Angiotensin II (Malhotra et al., 1999) that activate cellular responses known to
occur during hypertrophy in vivo. Although adult mice have a heart rate of over
600 beats per minute and an aorta that is 1.0 - 1.2 mm in diameter, they are a
valid model system to study both pressure-overload hypertrophy and heart
failure.
One of the most commonly used surgical intervention for pressure-overload induced
hypertrophy is coarction of the ascending aorta i.e. aortic banding. This system has been very well characterized and proven to be highly
reproducible with a low mortality rate of 10-20% or less in experienced hands.
Aortic banding is an excellent model system to evaluate the process of
development of left ventricular hypertrophy in response to hemodynamic stress.
Furthermore, after several months, a subset of animals progresses into heart
failure.
Studying changes in the gene expression profile at different time points
after the intervention will therefore give us valuable information on the
initial adaptation process and the transition from compensated hypertrophy to
failure. This information is critical for identifying potential novel
therapeutic targets in the future.
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Experimental Information
FVB wildtype mice were obtained from Charles River laboratories and operated
at 12 weeks of age.
Time points analyzed
Banded animals and sham-operated controls were sacrificed 1 hour, 4 hours, 24
hours, and 48 hours after the procedure to analyze immediate early and early
responses. In addition, the hypertrophic response was examined at one week and
eight weeks after the intervention during the chronic phase. Cardiac function
and the development of hypertrophy were assessed through echocardiographic
analysis and confirmed postmortem by determining the heart weight to body weight
ratio. The presence of pericardial effusion and ascites was recorded and lung
weight, liver weight, and tibial length were measured (see Physiology).
Gene expression changes in pressure-overload induced hypertrophy
It has been well documented that pressure overload rapidly activates a
program of immediate-early gene expression including c-fos, c-jun, jun-B, and
Egr-1. These changes occur within 15-30 minutes after aortic constriction. One
week after thoracic aortic banding, the hypertrophied ventricle is characterized
by a marked induction of both the atrial natriuretic factor (ANF) and the brain
natriuretic peptide (BNP) genes. These genes are excellent markers for the
induction of the hypertrophic response and serve as positive controls for the
gene expression analyses.
Operational Procedure
- Anesthesia: Pentobarbital 70mg/kg
- Post-op analgesia: Buprenex 0.1 mg/kg
After an adequate depth of anesthesia is attained the mouse is fixed in a supine position with tape. A
5-0 ligature is placed behind the front upper incisors and pulled taut so that the neck is slightly
extended. The tongue is retracted and held with forceps, and a 20-G i/v catheter is inserted into the
trachea. The catheter is then attached to the mouse ventilator (Model 687, Harvard Apparatus) via the
Y-shaped connector. Ventilation is performed with a tidal volume of 200ul and a respiratory rate of
133/min. 100% oxygen is provided to the inflow of the ventilator. Prior to the incision the chest is
disinfected with betadine solution, 70% ethyl alcohol, and 0.1ml of 0.1% lidocaine is introduced under the skin. The chest cavity
is opened by an incision of the left second intercostal space. Chest retractor is applied to facilitate the
view. The pericardial sac is opened and pulled
apart, the ascending portion of aorta is dissected from the surrounding tissues and a 7-0 silk suture is passed underneath of the ascending portion of the aorta and ligated against
a 25-G needle. The latter is immediately removed to provide a lumen with a stenotic aorta. Lungs are
overinflated, and the chest cavity, muscles and skin are closed layer by layer with 6-0 nylon and 6-0 absorbable (for muscles) sutures. The
duration of the whole procedure amounts to about 15-20 min.
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Page last modified: 26-Jan-2004
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