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

J Pharmacol Exp Ther. 2014 Apr;349(1):39-46. doi: 10.1124/jpet.113.210898. Epub 2014 Jan 15.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics
  • Adrenergic beta-1 Receptor Agonists / pharmacology
  • Animals
  • Animals, Newborn
  • Female
  • Gene Expression Regulation
  • Gene Knockdown Techniques*
  • Genetic Vectors
  • Isoproterenol / pharmacology
  • Male
  • Microarray Analysis
  • Myocardial Contraction* / drug effects
  • Myocardial Contraction* / genetics
  • Myocardium / cytology
  • Myocardium / metabolism*
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism*
  • RNA, Small Interfering / genetics
  • Rats
  • Rats, Inbred Lew
  • Rats, Wistar
  • Receptors, Adrenergic, beta-1 / genetics*
  • Tissue Culture Techniques
  • Tissue Engineering*


  • Adrenergic beta-1 Receptor Agonists
  • RNA, Small Interfering
  • Receptors, Adrenergic, beta-1
  • Isoproterenol