Construction of phospholamban antisense RNA recombinant adenoassociated virus vector and its effects in rat cardiomyocytes1
Abstract
Aim: 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.
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, a viral production positive control (rAAV-LacZ) and negative control were
performed. The recombinant viruses were used to transfect the cultured rat
cardiomyocytes. Site β-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.
Results: 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.
Conclusion: 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.
Keywords:
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.
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, a viral production positive control (rAAV-LacZ) and negative control were
performed. The recombinant viruses were used to transfect the cultured rat
cardiomyocytes. Site β-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.
Results: 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.
Conclusion: 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.