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


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.


CONCLUSIONS
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.
Li
Jiang
J
Department of Cardiology, First Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310003, China.
Hu
Shen-jiang
SJ
Sun
Jian
J
Zhu
Zhao-hui
ZH
Zheng
Xia
X
Wang
Guo-zhong
GZ
Yao
Yu-mei
YM
Chen
Nai-yun
NY
Zhao
Xiao-yan
XY
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics; D004622:Embryo, Mammalian; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney / Q000166:cytology; D007763:Lac Operon; D032383:Myocytes, Cardiac / Q000378:metabolism*; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D016372:RNA, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban