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(Circulation. 2000;101:2193.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Adenovirus-Based Phospholamban Antisense Expression as a Novel Approach to Improve Cardiac Contractile Dysfunction

Comparison of a Constitutive Viral Versus an Endothelin-1–Responsive Cardiac Promoter

Karin Eizema, PhD; Henry Fechner, DVM; Karel Bezstarosti, BSc; Sonja Schneider-Rasp, PhD; Arnoud van der Laarse, PhD; Haili Wang, MD; Heinz-Peter Schultheiss, MD; Wolfgang C. Poller, MD; Jos M. J. Lamers, PhD

From the Department of Biochemistry, Cardiovascular Research Institute COEUR, Erasmus University Rotterdam (K.E., K.B., J.M.J.L.), and the Department of Cardiology, Leiden University Medical Center (A.v.d.L.), Netherlands; and the Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Free University Berlin (H.F., S.S.-R., H.W., H.-P.S, W.C.P.), Germany.

Correspondence to J.M.J. Lamers, PhD, Department of Biochemistry, Cardiovascular Research Institute (COEUR), Faculty of Medicine and Health Sciences, Erasmus University, PO Box 1738, 3000 DR Rotterdam, Netherlands. E-mail lamers{at}bc1.fgg.eur.nl

Background—A decrease in sarcoplasmic reticulum Ca²⁺ pump (SERCA2) activity is believed to play a role in the impairment of diastolic function of the failing heart. Because the expression ratio of phospholamban (PL) to SERCA2 may be a target to improve contractile dysfunction, a PL antisense RNA strategy was developed under the control of either a constitutive cytomegalovirus (CMV) or an inducible atrial natriuretic factor (ANF) promoter. The latter is upregulated in hypertrophied and failing heart, allowing "induction-by-disease" gene therapy.

Methods and Results—Part of the PL cDNA was cloned in antisense and sense directions into adenovectors under the control of either a CMV (Ad5CMVPLas and Ad5CMVPLs, respectively) or ANF (Ad5ANFPLas and Ad5ANFPLs, respectively) promoter. Infection of cultured rat neonatal cardiomyocytes with Ad5CMVPLas reduced PL mRNA to 30±7% of baseline and PL protein to 24±3% within 48 and 72 hours, respectively. The effects were vector dose dependent. Ad5CMVPLas increased the Ca²⁺ sensitivity of SERCA2 and reduced the time to 50% recovery of the Ca²⁺ transient. A decrease of PL protein was also achieved by infection with Ad5ANFPLas, and the presence of the hypertrophic stimulus, endothelin-1, led to enhanced downregulation of PL. The adenovectors expressing PL sense RNA had no effect on any of the tested parameters.

Conclusions—Vector-mediated PL antisense RNA expression may become a feasible approach to modulate myocyte Ca²⁺ homeostasis in the failing heart. The inducible ANF promoter for the first time offers the perspective for induction-by-disease gene therapy, ie, selective expression of therapeutic genes in hypertrophied and failing cardiomyocytes.

Key Words: sarcoplasmic reticulum • calcium • endothelin • atrial natriuretic factor • adenovirus • gene therapy

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