Paradoxical Effects on Force Generation after Efficient β1-Adrenoceptor Knockdown in Reconstituted Heart Tissue

Stimulation of myocardial b1-adrenoceptors (AR) is a major mechanism that increases cardiac function. We investigated the functional consequences of genetic beta(1)-AR knockdown in three-dimensional engineered heart tissue (EHT). For beta(1)-AR knockdown, short interfering RNA (siRNA) sequences targeting specifically the beta(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 similar to 100%, and radioligand binding revealed 70% lower beta(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 beta-AR-stimulated conditions with isoprenaline (Delta fractional 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 beta(1)-AR knockdown itself determines the outcome. None of the regulated genes pointed to obvious offtarget effects to explain higher force generation. Moreover, compensational regulation of beta(2)-AR signaling or changes in prominent beta(1)-AR downstream targets could be ruled out. In summary, we show paradoxically higher force generation and isoprenaline responses after efficient beta(1)-AR knockdown in EHTs. Our findings 1) reveal an unexpected layer of complexity in gene regulation after specific beta(1)-AR knockdown rather than unspecific dysregulations through transcriptional interference, 2) challenge classic assumptions on the role of cardiac beta(1)-AR, and 3) may open up new avenues for b-AR loss-of-function research in vivo.