Atrial-Specific Gene Delivery Using an Adeno-Associated Viral Vector

Abstract

Rationale: Somatic overexpression in mice using an adeno-associated virus (AAV) as gene transfer vectors has become a valuable tool to analyze the roles of specific genes in cardiac diseases. The lack of atrial-specific AAV vector has been a major obstacle for studies into the pathogenesis of atrial diseases. Moreover, gene therapy studies for atrial fibrillation would benefit from atrial-specific vectors. Atrial natriuretic factor (ANF) promoter drives gene expression specifically in atrial cardiomyocytes.

Objective: To establish the platform of atrial specific in vivo gene delivery by AAV-ANF.

Methods and results: We constructed AAV vectors based on serotype 9 (AAV9) that are driven by the atrial-specific ANF promoter. Hearts from mice injected with AAV9-ANF-GFP (green fluorescent protein) exhibited strong and atrial-specific GFP expression without notable GFP in ventricular tissue. In contrast, similar vectors containing a cardiac troponin T promoter (AAV9-TNT4-GFP) showed GFP expression in all 4 chambers of the heart, while AAV9 with an enhanced chicken β-actin promoter (AAV-enCB-GFP) caused ubiquitous GFP expression. Next, we used Rosa26^mT/mG (membrane-targeted tandem dimer Tomato/membrane-targeted GFP), a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato before Cre-mediated excision, and membrane-targeted GFP after excision. AAV9-ANF-Cre led to highly efficient LoxP recombination in membrane-targeted tandem dimer Tomato/membrane-targeted green fluorescent protein mice with high specificity for the atria. We measured the frequency of transduced cardiomyocytes in atria by detecting Cre-dependent GFP expression from the Rosa26^mT/mG allele. AAV9 dose was positively correlated with the number of GFP-positive atrial cardiomyocytes. Finally, we assessed whether the AAV9-ANF-Cre vector could be used to induce atrial-specific gene knockdown in proof-of-principle experiments using conditional JPH2 (junctophilin-2) knockdown mice. Four weeks after AAV9-ANF-Cre injection, a strong reduction in atrial expression of JPH2 protein was observed. Furthermore, there was evidence for abnormal Ca²⁺ handling in atrial myocytes isolated from mice with atrial-restricted JPH2 deficiency.

Conclusions: AAV9-ANF vectors produce efficient, dose-dependent, and atrial-specific gene expression following a single-dose systemic delivery in mice. This vector is a novel reagent for both mechanistic and gene therapy studies on atrial diseases.

Keywords: animals; atrial fibrillation; atrial natriuretic factor; gene therapy; mice.

Figure 1.. AAV9-ANF-GFP induces atrial myocyte-specific GFP expression.

A, Schematic representation of the AAV transgenes used for these studies. ITR, I-terminal repeat; GFP, green fluorescent protein. B, Western blots showing GFP expression in atria of mice receiving different doses of AAV9-ANF-Cre (low: 5×10¹¹ GC; mid: 1×10¹² GC; high: 5×10¹² GC), or saline (Ctrl). C. Images of cardiac sections showing atrial-restricted GFP expression in mice treated with AAV9-ANF-GFP (1×10¹² GC). Mice receiving AAV9-TNT4-GFP and AAV9-enCB-GFP exhibited GFP expression in both atria and ventricles. D, Western blots showing GFP expression only in atrial tissue but not in ventricle or extra-cardiac tissues. E, Images of cardiac sections incubated with GFP antibody confirming atrial GFP expression in mice treated with AAV9-ANF-GFP, while both atria and ventricles expressed GFP in AAV9-TNT4-GFP treated mice. Scale bar, 100 μm.

Figure 2.. AAV9-ANF-Cre provides efficient LoxP recombination in atrial myocytes of mT/mG mice.

A, Schematic diagram of the AAV9-ANF-Cre vector used in the mT/mG mice. B, Schematic diagram of the mT/mG allele before and after Cre-mediated recombination. C, Representative images of hearts from mT/mG mice treated with AAV9-ANF-Cre and saline. Scale bar, 1.0 mm. D, Representative images of heart sections from mT/mG mice that were injected with different doses of AAV9-ANF-Cre (low: 5×10¹¹ GC; mid: 1×10¹² GC; high: 5×10¹² GC), or saline (Ctrl). Scale bar, 100 μm. E, Bar graph showing average percentages of GFP-positive cardiomyocytes. F, Representative images of heart sections from TAC mice pretreated with AAV9-ANF-Cre (1×10¹² GC) showing efficient Cre recombination in atrial myocytes resulting in GFP expression, while no GFP-positive cells were observed in ventricles. Scale bar, 100 μm. G, Echocardiography revealed reduced ejection fraction (EF) in mice 6 weeks after TAC. **p < 0.01; ***p < 0.001.

Figure 3.. AAV9-ANF-CRE mediated gene knockdown in atrial myocytes of NFATc1 floxed mice.

A, Schematic diagram of AAV9-ANF-Cre vector, and transgene in shJPH2 mice which contains a JPH2-silencing shRNA sequence downstream of a split U6 promoter, inactivate by a loxP-flanked neomycin stop element. B, AAV9-ANF-Cre administration to shJPH2 mice led to a reduction in atrial JPH2 protein levels assessed by western blotting, but C, while ventricular JPH2 levels remained unaltered. D, Western blots showing reduced atrial, and E, ventricular JPH2 protein levels in shJPH2 mice treated with AAV9-TNT4-Cre. N, number of mice. p<0.05, **p<0.01.

Figure 4.. Abnormal SR Ca²⁺ handling in atrial myocytes from shJPH2 mice injected with AAV9-ANF-Cre.

A, Representative tracings of Ca²⁺ transients after 1-Hz pacing in atrial myocytes from shJPH2 mice. Caffeine induced SR Ca²⁺ dump. B, Quantification of Ca²⁺ transient amplitude (CaT), and C, total SR Ca²⁺ content (SR load). D, Confocal microscopy line scans revealing Ca²⁺ sparks in atrial myocytes from shJPH2 mice. E, Bar graphs showing average Ca²⁺ spark frequency (CaSpF), and F, CaSpF normalized to SR Ca²⁺ load. *P<0.05; *** P<0.001.