Construction of phospholamban antisense RNA recombinant adeno-associated virus vector and its effects in rat cardiomyocytes1

Abstract

To construct a recombinant adeno-associated virus (rAAV) vector containing gene encoding phospholamban antisense RNA (asPLB), and analyse its effect on expression of PLB, expression and activity of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and the change of intracellular free Ca2+ concentration ([Ca2+]i) in rat cardiomyocytes. The target gene encoding PLB antisense RNA was inserted inversely into the adeno-associated virus plasmid pAAV-MCS digested by corresponding restricted endonuclease enzyme. The recombinant plasmid and pAAV-RC and pHelper were co-transfected into 293 cell. At the same time, a viral production positive control (rAAV-LacZ) and negative control were performed. The recombinant viruses were used to transfect the cultured rat cardiomyocytes. Site beta-Galactosidase staining were performed to observe the transfer efficiency. Reverse transcription-PCR and Western blot were used to determine the mRNA and protein expression of PLB and SERCA. The activity of SERCA and the [Ca2+]i were measured. The rAAV vectors were constructed successfully and were transfected into rat cardiomyocytes effectively. The PLB mRNA and protein expression were reduced in rat cardiomyocytes transfected by rAAV-asPLB compared with controls. The activity of SERCA was increased. In rest state, the level of [Ca2+]i in the rAAV-asPLB transfected group decreased. The level of [Ca2+]i increased when induced by isoproterenol. AAV-asPLB vector was constructed successfully, which disrupted the expression of PLB, enhanced the activity of SERCA, reduced the resting [Ca2+]i, and improved the cardiac function.