Aim:
Assess the effect of ACE2 overexpression
in vivo
on heart function and blood pressure. Identify an efficient cardiac gene delivery vector by comparison and optimisation of adeno-associated virus 6 (AAV6) and AAV9 mediated gene delivery to myocardium
in vivo
in SHRSPs.
Methods:
We administered a single intravenous injection of AAV6CMVlacZ or AAV9CMVlacZ at 3 doses (2x10
11
, 1.5x10
12
and 3x10
12
vp/rat) into 6 week old SHRSP. Animals were sacrificed 14 days post delivery and tissues stained for β-galactosidase (β-gal) expression which was confirmed by immunohistochemistry (IHC). Quantitative PCR compared the presence of lacZ containing genomes between tissues and animals. To assess the effect of ACE2 overexpression in SHRSP, 4 groups of animals (n = 6/group) were included in the study; PBS, Enalapril, AAV6-alkaline phosphatase (control reporter gene) and AAV6-ACE2. Blood pressure was monitored by tail cuff and cardiac function assessed by echocardiography (ECHO). Cardiac structure was assessed by haematoxylin and eosin (H&E) staining and cardiac fibrosis evaluated by Masson’s trichrome and picrosirius red.
Results:
AAV6-mediated gene transfer was high in heart and skeletal muscle and a dose-dependent response was observed. β-gal staining and IHC confirmed transgene expression throughout the musculature but not within other tissues (kidney, liver, spleen and lung). Whilst AAV9 mediated approximately 10 fold (for highest dose) less gene transfer to the heart than AAV6, levels were comparable in skeletal muscle. However, the AAV9 vector accumulated in the kidneys. ACE2 was overexpressed selectively in the AAV6-ACE2-injected animals. ECHO showed substantial systolic dysfunction in ACE2-injected SHRSP’s compared to controls, and H&E revealed abnormal cardiac structure. Masson’s trichrome and picrosirius red indicated severe cardiac fibrosis. Blood pressure was significantly lower (p<0.001) in the ACE2 group in weeks 9, 10 and 11 post infusion compared to PBS and control vector infused animals.
Conclusions:
AAV6 exhibited a more favourable profile for cardiac gene delivery than AAV9, and represents a useful tool for studying mechanisms of cardiovascular disease. Overexpression of ACE2 in SHRSP myocardium led to severe cardiac fibrosis.
In Vivo Cardiac Gene Transfer of Kv4.3 Abrogates the Hypertrophic Response in Rats After Aortic Stenosis
