Abstract
Stimulation of myocardial β1-adrenoceptors (AR) is a major mechanism that increases cardiac function. We investigated the functional consequences of genetic β1-AR knockdown in three-dimensional engineered heart tissue (EHT). For β1-AR knockdown, short interfering RNA (siRNA) sequences targeting specifically the β1-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 β1-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 β1-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 β2-AR signaling or changes in prominent β1-AR downstream targets could be ruled out. In summary, we show paradoxically higher force generation and isoprenaline responses after efficient β1-AR knockdown in EHTs. Our findings 1) reveal an unexpected layer of complexity in gene regulation after specific β1-AR knockdown rather than unspecific dysregulations through transcriptional interference, 2) challenge classic assumptions on the role of cardiac β1-AR, and 3) may open up new avenues for β-AR loss-of-function research in vivo.
Footnotes
- Received November 4, 2013.
- Accepted January 13, 2014.
T.E. and A.E.-A. contributed equally to this work.
This study was supported by the Deutsche Forschungsgemeinschaft [DFG FOR 604 (to T.E. and A.E.A.), DFG EL 270/5-1 and SFB 1002 TP-A02 (to A.E.A.)], Deutsche Herzstiftung (to A.E.A.), and DZHK [German Center for Cardiovascular Research (to T.E. and A.E.A.)].
↵This article has supplemental material available at jpet.aspetjournals.org.
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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