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A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca(2+) Uptake into the Sarcoplasmic Reticulum

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 Ca(2+) cycling and contractile dysfunction. Cardiac delivery of shPLB, howeve...

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Autores principales: Größl, Tobias, Hammer, Elke, Bien-Möller, Sandra, Geisler, Anja, Pinkert, Sandra, Röger, Carsten, Poller, Wolfgang, Kurreck, Jens, Völker, Uwe, Vetter, Roland, Fechner, Henry
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3966758/
https://www.ncbi.nlm.nih.gov/pubmed/24670775
http://dx.doi.org/10.1371/journal.pone.0092188
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author Größl, Tobias
Hammer, Elke
Bien-Möller, Sandra
Geisler, Anja
Pinkert, Sandra
Röger, Carsten
Poller, Wolfgang
Kurreck, Jens
Völker, Uwe
Vetter, Roland
Fechner, Henry
author_facet Größl, Tobias
Hammer, Elke
Bien-Möller, Sandra
Geisler, Anja
Pinkert, Sandra
Röger, Carsten
Poller, Wolfgang
Kurreck, Jens
Völker, Uwe
Vetter, Roland
Fechner, Henry
author_sort Größl, Tobias
collection PubMed
description 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.
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spelling pubmed-39667582014-03-31 A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca(2+) Uptake into the Sarcoplasmic Reticulum Größl, Tobias Hammer, Elke Bien-Möller, Sandra Geisler, Anja Pinkert, Sandra Röger, Carsten Poller, Wolfgang Kurreck, Jens Völker, Uwe Vetter, Roland Fechner, Henry PLoS One Research Article 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile. Public Library of Science 2014-03-26 /pmc/articles/PMC3966758/ /pubmed/24670775 http://dx.doi.org/10.1371/journal.pone.0092188 Text en © 2014 Größl et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Größl, Tobias
Hammer, Elke
Bien-Möller, Sandra
Geisler, Anja
Pinkert, Sandra
Röger, Carsten
Poller, Wolfgang
Kurreck, Jens
Völker, Uwe
Vetter, Roland
Fechner, Henry
A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca(2+) Uptake into the Sarcoplasmic Reticulum
title A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca(2+) Uptake into the Sarcoplasmic Reticulum
title_full A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca(2+) Uptake into the Sarcoplasmic Reticulum
title_fullStr A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca(2+) Uptake into the Sarcoplasmic Reticulum
title_full_unstemmed A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca(2+) Uptake into the Sarcoplasmic Reticulum
title_short A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca(2+) Uptake into the Sarcoplasmic Reticulum
title_sort novel artificial microrna expressing aav vector for phospholamban silencing in cardiomyocytes improves ca(2+) uptake into the sarcoplasmic reticulum
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3966758/
https://www.ncbi.nlm.nih.gov/pubmed/24670775
http://dx.doi.org/10.1371/journal.pone.0092188
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