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.
BACKGROUND
A decrease in sarcoplasmic reticulum Ca(2+) 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.


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 Ca(2+) sensitivity of SERCA2 and reduced the time to 50% recovery of the Ca(2+) 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 Ca(2+) 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.
Eizema
K
K
Department of Biochemistry, Cardiovascular Research Institute COEUR, Erasmus University Rotterdam, The Netherlands.
Fechner
H
H
Bezstarosti
K
K
Schneider-Rasp
S
S
van der Laarse
A
A
Wang
H
H
Schultheiss
H P
HP
Poller
W C
WC
Lamers
J M
JM
D000256:Adenoviridae; D000818:Animals; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D000252:Calcium-Transporting ATPases / Q000235:genetics; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006321:Heart / Q000502:physiology*; D009200:Myocardial Contraction / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D019332:Endothelin-1; D016376:Oligonucleotides, Antisense; D000099211:Phospholamban