title: Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.
abstract: Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network.
authors.affiliation: Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Schillingallee 69, 18057 Rostock, Germany. Electronic address: heiko.lemcke@uni-rostock.de.
authors.forename: Heiko
authors.initials: H
authors.lastname: Lemcke
authors.affiliation: Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Schillingallee 69, 18057 Rostock, Germany.
authors.forename: Janine
authors.initials: J
authors.lastname: Peukert
authors.affiliation: Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Schillingallee 69, 18057 Rostock, Germany.
authors.forename: Natalia
authors.initials: N
authors.lastname: Voronina
authors.affiliation: Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Schillingallee 69, 18057 Rostock, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Skorska
authors.affiliation: Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Schillingallee 69, 18057 Rostock, Germany.
authors.forename: Gustav
authors.initials: G
authors.lastname: Steinhoff
authors.affiliation: Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Schillingallee 69, 18057 Rostock, Germany. Electronic address: robert.david@med.uni-rostock.de.
authors.forename: Robert
authors.initials: R
authors.lastname: David
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002450:Cell Communication; D036681:Fluorescence Recovery After Photobleaching; D017629:Gap Junctions / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods; D032383:Myocytes, Cardiac / Q000378:metabolism*; D034443:RNA Transport; D016372:RNA, Antisense / Q000378:metabolism*
chemical_list: D035683:MicroRNAs; D016372:RNA, Antisense
keywords: Connexins; Gap junctions; Intercellular communication; Neonatal cardiomyocytes; Small noncoding RNA; miRNA transfer