The Effects of Doxycycline on Cardiac and
Non-cardiac
Beta2AR Expression
To gauge the expression of Beta2 adrenergic
receptors (Beta2ARs) in the hearts of conditional
cardiac specific Beta2AR knockout mice (Beta2KO) after
doxycycline administration.
Beta1ARs and Beta2Rs: Previous studies
using conventional Beta2KO mice have provided evidence
that Beta2AR activation protects the murine heart
during continuous Beta1AR stimulation. This finding
is significant for clinical medicine because continuous
Beta1AR stimulation has been shown to mediate much
of the myocardial injury that occurs in congestive
heart failure. Why Beta2AR activation is protective
during continuous Beta1AR stimulation is unclear,
but may be related to the Beta2ARs ability to couple
to inhibitory G protein (which Beta1ARs cannot do).
Beta1ARs and Beta2ARs are otherwise very similar receptors
and are activated by the same ligands.
Conditional versus Conventional Beta2KO
Mice: The previous studies demonstrating that Beta2AR
activation protects the murine heart during continuous
Beta1AR stimulation were performed using conventional
Beta2KO mice. These animals develop in utero and postnatally
without Beta2ARs, suggesting that the animals may
develop means to compensate for the absence of Beta2ARs.
In addition, the Beta2AR disruption in these animals
is not cardiac specific. Therefore, it was not clear
from the previous studies whether cardiac or non-cardiac
Beta2ARs conveyed the observed protective effects.
TetR and Cre/loxP Conditional Knockout:
I will study mice with a conditional cardiac specific
Beta2AR disruption. The animals were engineered such
that cardiac specific expression of a reverse transcriptional
transactivator results in cre expression and disruption
of the Beta2AR gene only in the presence of an inducer,
doxycycline. In theory, we should be able to disrupt
only cardiac Beta2AR expression at any time in these
adult animals by administering doxycycline.
My Project ñ Analysis of the
Conditional Cardiac Specific Knockout: I will assess
cardiac and non-cardiac Beta2AR expression in the
conditional Beta2KO mice after increasing doses of
doxycycline. I will use the following approach:
1. I will study four groups of animals
(5 animals in each group): Wild Type, Conventional
Beta2KO, Untreated Conditional Beta2KO, and Treated
Conditional Beta2KO.
2. For treated Conditional Beta2KO mice
I will administer doxycycline (from 0.5 mg/day infusion
to 6 mg/day infusion) for 5 days using subcutaneous
infusion pumps.
3. After sacrificing the animals, I
will harvest RNA from their hearts and tails.
4. I will produce cDNA using reverse
transcription polymerase chain reaction (rtPCR), then
perform conventional PCR using primers designed to
amplify a segment of the Beta2AR. I will also perform
PCR using primers designed to amplify a segment of
the neomycin cassette that was inserted within the
floxed segment immediately upstream from the Beta2AR
gene in conditional Beta2KO mice.
I will work with another student and
a research associate in the host laboratory. With
the exception of rtPCR, I learned to perform all of
the proposed techniques last summer while working
in the host laboratory.
Activities
Associated with Award
Meeting/Poster Presentation:
Marissa Patterson, Jim Wong, M.D.,
Ashwin Murthy, March 2007. Beta-Adrenergic Receptors
(βAR): Role in Modulating the Host Immune
Response. Seminars in Anesthesia, Perioperative Medicine
and Pain.
