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 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 AdSCMVPLas 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. AdSCMVPLas 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-l, led to enhanced downregulation of FL. 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.