Robust adenoviral and adeno-associated viral gene transfer to the in vivo murine heart: application to study of phospholamban physiology.
BACKGROUND
Viral gene transfer to the whole heart in vivo has been achieved in several mammalian species but remained difficult to accomplish in murine hearts. We postulated that a key impediment derives from the use of proximal aortic occlusion during virus injection, because this eliminates coronary perfusion gradients in mice as aortic root and left ventricle pressures equalize.


RESULTS
Pressure-volume analysis confirmed these mechanics. In contrast, descending aortic occlusion with whole-body cooling (20 degrees C) preserved transmyocardial perfusion gradients and allowed for sustained (>10-minute) dwell times in an upper-body perfusion circuit. This approach yielded robust cardiac transfection with adenovirus (AdV) and adeno-associated virus (AAV) injected into the left ventricle cavity or more simply via a central vein. Cardio-specific expression was achieved with a myocyte-specific promotor. Optimal AdV transfection required 9-minute aortic occlusion, versus 5-minute occlusion for AAV. Using this method, we examined the in vivo function of phospholamban (PLB) by stably transfecting PLB-null mice with AAV encoding PLB (AAV(PLB)). AAV(PLB) restored PLB protein to near control levels that colocalized with SERCA2A in cardiomyocytes. At baseline, PLB-null hearts exhibited enhanced systolic and diastolic function, but frequency-dependent reserve was blunted versus wild-type controls. These properties, particularly the frequency response, returned toward control 3 months after AAV(PLB) transfection.


CONCLUSIONS
The new simplified approach for murine whole-heart viral transfection should assist molecular physiology studies.
Champion
Hunter C
HC
Division of Cardiology, Department of Medicine, 600 N Wolfe St, Halsted 500, Johns Hopkins Hospital, Baltimore, Md 21287, USA. hchampi@jhmi.edu
Georgakopoulos
Dimitrios
D
Haldar
Saptarsi
S
Wang
Lili
L
Wang
Yibin
Y
Kass
David A
DA
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001011:Aorta / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis; D002469:Cell Separation; D003326:Coronary Circulation; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D006321:Heart / Q000502:physiology* / Q000821:virology; D051379:Mice; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013997:Time Factors; D016277:Ventricular Function, Left / Q000235:genetics / Q000502:physiology; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D001616:beta-Galactosidase; D000099211:Phospholamban