title: A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.
abstract: In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Gröβl
authors.affiliation: Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Hammer
authors.affiliation: Department of Pharmacology, Center of Drug Absorption and Transport, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Bien-Möller
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Geisler
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Pinkert
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Röger
authors.affiliation: Department of Cardiology & Pneumology, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Poller
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Kurreck
authors.affiliation: Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Völker
authors.affiliation: Institute of Clinical Pharmacology & Toxicology, Charité - Universitätsmedizin Berlin, Campus Charité Mitte, Berlin, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Vetter
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors.forename: Henry
authors.initials: H
authors.lastname: Fechner
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D000229:Dependovirus / Q000378:metabolism*; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000473:pathology; D035683:MicroRNAs / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012519:Sarcoplasmic Reticulum; D014161:Transduction, Genetic; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D035683:MicroRNAs; D012333:RNA, Messenger; D000099211:Phospholamban