Assessment of Gene Transfer in Rat Hearts
Jesus Jimenez, Jeff Drumm, BS; Aziz N. Ander BS; Mei Hua Gao, PhD;
David M. Roth, MD, PhD; H. Kirk Hammond, MD
Introduction
Congestive heart failure is an increasingly predominant cause of mortality.
Although current methods exist to help treat this condition, gene therapy is an alternative
method being addressed.
Cardiac gene therapy involves the introduction of a potentially therapeutic gene
into the heart via a vector resulting in the expression of the desired protein. Our lab is
studying the effects of expressing adenylyl cyclase isoform VI (ACVI). AC is a molecule
in the -adrenergic receptor (AR) signaling cascade, which catalyzes the conversion of
ATP to cAMP. Cardiac-directed expression of ACVI in mice increased the levels of
cAMP and attenuates the decrease in function associated with cardiomyopathy (Roth et
al, 1999).
The objective of this study was to assess gene transfer to the rat heart via indirect
intracoronary delivery of an adenovirus encoding enhanced green fluorescence protein
(EGFP). Cells infected with EGFP appear green under fluorescence. Antibody staining
and deconvolution microscopy can also be used to determine which cell types express the
protein. Our hypothesis was that genes delivered via adenovirus vectors will be
expressed primarily in cardiac myocytes.
Methods
The vector used is a replication-deficient, serotype 5 adenovirus in which the
E1A/E1B genes were replaced by EGFP or ACVI (Adv.EGFP or Adv.ACVI). Animal use
was in accordance with National Institutes of Health and institutional guidelines.
Adv/EGFP or Adv/ACVI (2.5x1011 viral particles) was injected into the proximal aortic
root during a cross-clamp of the aorta and the pulmonary artery. Ten days after vector
delivery , the animals were killed. The heart was removed and perfusion-fixed with 4%
paraformaldehyde. The heart was initially viewed under fluorescent microscopy to
determine the extent of gene transfer. Western blot analysis was also used to detect
presence of EGFP or ACVI. The heart was then paraffin embedded, sliced, and mounted
on slides to be stained with antibodies and viewed using deconvolution microscopy.
Hearts that underwent the gene delivery procedure but were injected with saline were
used as controls.
Results
Fluorescent cardiac myocytes were observed in infected hearts (Figure 1). Only
background autofluorescence was observed in control tissue (not shown). The Western
blot analysis of transfected myocytes showed clear bands for EGFP appearing at 38kDa
(Figure 2). Antibody staining and deconvolution microscopy also revealed the presence
of EGFP in cardiomyocytes of infected tissue but not in control tissue (Figure 3). The
blue stains in Figure 3 are nuclei and the red stains are myocytes expressing EGFP.
Figure 1
Figure 2
Figure 3
Discussion
Cardiac-directed expression of ACVI has been shown to increase survival in
cardiomyopathy in mouse models (Roth et al, 2002). Indirect intracoronary gene delivery
in rats would be a way to assess the effects of transient ACVI expression. We used EGFP
to determine the extent of gene transfer in the rat heart and which cell types became
infected. Fluorescent microscopy and antibody staining followed by deconvolution
microscopy showed that cardiac myocytes in infected heart tissue were expressing EGFP.
We have shown that indirect intracoronary delivery is a viable method of gene transfer to
the rat heart and may be used to assess therapeutic effects of functional genes such as
ACVI in the future.