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


 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.  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.  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.  AAV9-ANF vectors produce efficient, dose-dependent, and atrial-specific gene expression following a single-dose systemic delivery in mice. 
AAV
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.  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.  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.  AAV9-ANF vectors produce efficient, dose-dependent, and atrial-specific gene expression following a single-dose systemic delivery in mice. 
AAV9
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 Rosa26mT/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.  We measured the frequency of transduced cardiomyocytes in atria by detecting Cre-dependent GFP expression from the Rosa26mT/mG allele.  AAV9 dose was positively correlated with the number of GFP-positive atrial cardiomyocytes. 
GFP
Next, we used Rosa26mT/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 Rosa26mT/mG allele.  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. 
Cre
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. 
LoxP