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

Background—A decrease in sarcoplasmic reticulum Ca21 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 3067 % of baseline and PL protein to 2463 % within 48 and 72 hours, respectively. The effects were vector dose dependent. Ad5CMVPLas increased the Ca21 sensitivity of SERCA2 and reduced the time to 50 % recovery of the Ca21 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 Ca21 homeostasis in the failing heart. The inducible ANF promoter for the first time offers the perspective fo