Paradoxical effects on force generation after efficient β1-adrenoceptor knockdown in reconstituted heart tissue
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Paradoxical effects on force generation after efficient β1-adrenoceptor knockdown in reconstituted heart tissue. / Neuber, Christiane; Müller, Oliver J; Hansen, Felix ; Eder, Alexandra; Witten, Anika; Rühle, Frank; Stoll, Monika; Katus, Hugo A; Eschenhagen, Thomas; El-Armouche, Ali.
in: J PHARMACOL EXP THER, Jahrgang 349, Nr. 1, 01.04.2014, S. 39-46.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Paradoxical effects on force generation after efficient β1-adrenoceptor knockdown in reconstituted heart tissue
AU - Neuber, Christiane
AU - Müller, Oliver J
AU - Hansen, Felix
AU - Eder, Alexandra
AU - Witten, Anika
AU - Rühle, Frank
AU - Stoll, Monika
AU - Katus, Hugo A
AU - Eschenhagen, Thomas
AU - El-Armouche, Ali
N1 - Felix Hansen als interne Person anlegen, ansonsten ok
PY - 2014/4/1
Y1 - 2014/4/1
N2 - 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.
AB - 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.
KW - Adenoviridae
KW - Adrenergic beta-1 Receptor Agonists
KW - Animals
KW - Animals, Newborn
KW - Female
KW - Gene Expression Regulation
KW - Gene Knockdown Techniques
KW - Genetic Vectors
KW - Isoproterenol
KW - Male
KW - Microarray Analysis
KW - Myocardial Contraction
KW - Myocardium
KW - Myocytes, Cardiac
KW - RNA, Small Interfering
KW - Rats
KW - Rats, Inbred Lew
KW - Rats, Wistar
KW - Receptors, Adrenergic, beta-1
KW - Tissue Culture Techniques
KW - Tissue Engineering
U2 - 10.1124/jpet.113.210898
DO - 10.1124/jpet.113.210898
M3 - SCORING: Journal article
C2 - 24431469
VL - 349
SP - 39
EP - 46
JO - J PHARMACOL EXP THER
JF - J PHARMACOL EXP THER
SN - 0022-3565
IS - 1
ER -