title: Paradoxical effects on force generation after efficient β1-adrenoceptor knockdown in reconstituted heart tissue.
abstract: Stimulation of myocardial β(1)-adrenoceptors (AR) is a major mechanism that increases cardiac function. We investigated the functional consequences of genetic β(1)-AR knockdown in three-dimensional engineered heart tissue (EHT). For β(1)-AR knockdown, short interfering RNA (siRNA) sequences targeting specifically the β(1)-AR (shB1) and a scrambled control (shCTR) were subcloned into a recombinant adeno-associated virus (AAV)-short hairpin RNA (shRNA) expression system. Transduction efficiency was ∼100%, and radioligand binding revealed 70% lower β(1)-AR density in AAV6-shB1-transduced EHTs. Force measurements, performed over the culture period of 14 days, showed paradoxically higher force generation in AAV6-shB1 compared with shCTR under basal (0.19 ± 0.01 versus 0.13 ± 0.01 mN) and after β-AR-stimulated conditions with isoprenaline (Δfractional shortening: 72 ± 5% versus 34 ± 4%). Large scale gene expression analysis revealed that AAV6-shCTR compared with nontransduced EHTs showed only few differentially regulated genes (<20), whereas AAV6-shB1 induced marked changes in gene expression (>250 genes), indicating that β(1)-AR knockdown itself determines the outcome. None of the regulated genes pointed to obvious off-target effects to explain higher force generation. Moreover, compensational regulation of β(2)-AR signaling or changes in prominent β(1)-AR downstream targets could be ruled out. In summary, we show paradoxically higher force generation and isoprenaline responses after efficient β(1)-AR knockdown in EHTs. Our findings 1) reveal an unexpected layer of complexity in gene regulation after specific β(1)-AR knockdown rather than unspecific dysregulations through transcriptional interference, 2) challenge classic assumptions on the role of cardiac β(1)-AR, and 3) may open up new avenues for β-AR loss-of-function research in vivo.
authors.affiliation: Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Germany, and DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Germany (C.N., F.C.H., A.E., T.E.); Internal Medicine III, University Hospital Heidelberg, Germany, and DZHK (German Center for Cardiovascular Research), partner site Heidelberg, Germany (O.J.M, H.A.K.); Leibniz-Institute for Arteriosclerosis Research, University of Münster, Münster, Germany (A.W., F.R., M.S.); and Institute of Pharmacology, University Medical Center Göttingen, Göttingen, Germany, and DZHK (German Center for Cardiovascular Research), partner site Göttingen, Germany, and Department of Pharmacology, University of Technology Dresden, Dresden, Germany (A.E.-A.).
authors.forename: Christiane
authors.initials: C
authors.lastname: Neuber
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Müller
authors.forename: Felix C
authors.initials: FC
authors.lastname: Hansen
authors.forename: Alexandra
authors.initials: A
authors.lastname: Eder
authors.forename: Anika
authors.initials: A
authors.lastname: Witten
authors.forename: Frank
authors.initials: F
authors.lastname: Rühle
authors.forename: Monika
authors.initials: M
authors.lastname: Stoll
authors.forename: Hugo A
authors.initials: HA
authors.lastname: Katus
authors.forename: Thomas
authors.initials: T
authors.lastname: Eschenhagen
authors.forename: Ali
authors.initials: A
authors.lastname: El-Armouche
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D058665:Adrenergic beta-1 Receptor Agonists / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D005260:Female; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D046228:Microarray Analysis; D009200:Myocardial Contraction / Q000187:drug effects / Q000235:genetics; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew; D017208:Rats, Wistar; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics*; D046509:Tissue Culture Techniques; D023822:Tissue Engineering
chemical_list: D058665:Adrenergic beta-1 Receptor Agonists; D034741:RNA, Small Interfering; D018342:Receptors, Adrenergic, beta-1; D007545:Isoproterenol