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 target potentially therapeutic gene into the heart via a vector resulting in the over-expression of the desired protein. Our lab is studying the effects of over-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 Over-expression of ACVI in mice increased the levels of cAMP and attenuatesd the decrease in function associated with cardiomyopathy (Roth et al, 1999).

The objective of this study wasis to assess gene transfer to the rat heart via indirect intracoronary delivery of an adenovirus encoding carrying enhanced green fluorescence protein (EGFP) gene. Cells infected with EGFP will appear emit green color under fluorescence. Antibody staining and deconvolution microscopy can also be used to determine which cell types express the protein. Our hypothesis wasis that genes delivered via adenovirus viral 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 roof root during a cross-clamp of the aorta and the pulmonary artery. Ten days after post-vector delivery transfection, the animals were killedeuthanized. 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 Over-expression of ACVI has been shown to increase survival in of cardiomyopathy in mouse models (Roth et al, 2002). Indirect intracoronary gene delivery in rats would be a way to assess the effects of transient ACVIover-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 over-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.