Effects of phospholamban antisense RNA on SERCA and CaI in rat cardiomyocytes using recombinant adeno-associated virus vector

摘要

AIM To construct a recombinant adeno-associated virus (rAAV) vector containing gene encoding phospholamban antisense RNA (asPLB), as well as analyse its effect on expression of PLB, expression and activity of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and the chang of intracellular free Ca2+ concentration ([Ca2+]I) in rat cardiomyocytes. METHODS 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, performing a viral production positive control (rAAV-LacZ) and negative control. The recombinant viruses were used to transfect the cultured rat cardiomyocytes. Site β -Galactosidase Staining were performed to observe the transfer efficiency . RT-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. RESULTS The rAAV vectors are constructed successfully and transfected into rat cardiolmyocytes effectively. The PLB mRNA and protein expression are reduced in rat cardiomyocytes which transfected by rAAV-asPLB compared with controls. The activity of SERCA was increased. In rest state, the level of [Ca2+]I in rAAV-asPLB transfected group was decreased. The level of [Ca2+]I was increased when induced by isoproterenol, and the duration of the diastolic Ca2+ was reduced. CONCLUSION rAAV-asPLB vector was constructed successfully and disrupts the expression of PLB, enhances the activity of SERCA,. Reduced the resting [Ca2+]I and the duration of diastolic Ca2+.