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Paradoxical effects on force generation after efficient β1-adrenoceptor knockdown in reconstituted heart tissue.

Authors
  • Neuber, Christiane1
  • Müller, Oliver J
  • Hansen, Felix C
  • Eder, Alexandra
  • Witten, Anika
  • Rühle, Frank
  • Stoll, Monika
  • Katus, Hugo A
  • Eschenhagen, Thomas
  • El-Armouche, Ali
  • 1 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.). , (Germany)
Type
Published Article
Journal
Journal of Pharmacology and Experimental Therapeutics
Publisher
American Society for Pharmacology & Experimental Therapeutics
Publication Date
Apr 01, 2014
Volume
349
Issue
1
Pages
39–46
Identifiers
DOI: 10.1124/jpet.113.210898
PMID: 24431469
Source
Medline
License
Unknown

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.

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