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(Circulation. 2003;108:2790.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Robust Adenoviral and Adeno-Associated Viral Gene Transfer to the In Vivo Murine Heart

Application to Study of Phospholamban Physiology

Hunter C. Champion, MD, PhD; Dimitrios Georgakopoulos, PhD; Saptarsi Haldar, MD; Lili Wang, PhD; Yibin Wang, PhD; David A. Kass, MD

From the Division of Cardiology, Department of Medicine (H.C.C., D.G., S.H., D.A.K.), Johns Hopkins Hospital, Baltimore, Md; Institute for Human Gene Therapy (L.W.), University of Pennsylvania, Philadelphia, Pa; and University of Maryland School of Medicine (Y.W.), Baltimore, Md.

Correspondence to Hunter C. Champion, MD, PhD, Division of Cardiology, Department of Medicine, 600 N Wolfe St, Halsted 500, Johns Hopkins Hospital, Baltimore, MD 21287. E-mail hchampi{at}jhmi.edu

Received February 18, 2003; de novo received May 6, 2003; revision received July 9, 2003; accepted August 4, 2003.

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.

Methods and Results— Pressure-volume analysis confirmed these mechanics. In contrast, descending aortic occlusion with whole-body cooling (20°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.

Key Words: gene therapy • viruses • aorta • cardioplegia • contractility

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