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

AbstractAim: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.

[1]  Gui-mei Zhang,et al.  Down-regulation of survivin expression reversed multidrug resistance in adriamycin-resistant HL-60/ADR cell line. , 2003, Acta pharmacologica Sinica.

[2]  R. Hajjar,et al.  Gene therapy for the treatment of heart failure--calcium signaling. , 2003, Seminars in thoracic and cardiovascular surgery.

[3]  Jirun Peng,et al.  [Construction, packaging and titration of recombinant adeno-associated virus vectors contaning antitumor genes]. , 2002, Zhonghua yi xue za zhi.

[4]  Harvard Medical School,et al.  Targeting Phospholamban by Gene Transfer in Human Heart Failure , 2002, Circulation.

[5]  E. Kranias,et al.  Phospholamban and cardiac contractile function. , 2000, Journal of molecular and cellular cardiology.

[6]  S. Teramoto,et al.  Recombinant adeno-associated virus vectors efficiently transduce foreign gene into bovine aortic endothelial cells: comparison with adenovirus vectors. , 2000, Japanese journal of pharmacology.

[7]  B. Hoit,et al.  Cardiac-specific Overexpression of a Superinhibitory Pentameric Phospholamban Mutant Enhances Inhibition of Cardiac Functionin Vivo * , 2000, The Journal of Biological Chemistry.

[8]  W. Lew,et al.  Effects of mutant and antisense RNA of phospholamban on SR Ca(2+)-ATPase activity and cardiac myocyte contractility. , 1999, Circulation.

[9]  G. Qiao,et al.  Antagonistic effects of berbamine on [Ca2+]i mobilization by KCl, norepinephrine, and caffeine in newborn rat cardiomyocytes. , 1999, Zhongguo yao li xue bao = Acta pharmacologica Sinica.

[10]  E. Svensson,et al.  Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors. , 1999, Circulation.

[11]  K. Clark,et al.  A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors. , 1996, Gene therapy.

[12]  G. Dorn,et al.  Cardiac-specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanics in transgenic mice. , 1996, The Journal of clinical investigation.

[13]  K. Kjeldsen,et al.  Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes , 1995, Basic Research in Cardiology.

[14]  T. Doetschman,et al.  Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. , 1994, Circulation research.

[15]  B. Carter,et al.  Adeno-associated virus vectors , 1992, Current Biology.

[16]  I. Grupp,et al.  The relative phospholamban and SERCA2 ratio: a critical determinant of myocardial contractility , 2004, Basic Research in Cardiology.