Paradoxical Effects on Force Generation after Efficient β1-Adrenoceptor Knockdown in Reconstituted Heart Tissue
Paradoxical Effects on Force Generation after Efficient β1-Adrenoceptor Knockdown in Reconstituted Heart Tissue
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DOI：
10.1124/jpet.113.210898
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发表时间：
2014-04-01
影响因子：
3.5
通讯作者：
El-Armouche, Ali
中科院分区：
文献类型：
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作者：
Neuber, Christiane;Mueller, Oliver J.;El-Armouche, Ali
心肌b1肾上腺素受体（AR）的刺激是增加心功能的主要机制。我们研究了基因β (1)-AR敲低在三维工程心脏组织（EHT）中的功能后果。为了敲低β (1)-AR，我们将特异性靶向β (1)-AR （shB1）和一个混乱对照（shCTR）的短干扰RNA （siRNA）序列亚克隆到重组腺相关病毒(AAV)-短发夹RNA （shRNA）表达系统中。转导效率接近100%，辐射配体结合显示aav6 - shb1转导的eht β (1)-AR密度降低70%。在14天的培养期间进行的力测量显示，与基础（0.19 +/- 0.01 mN对0.13 +/- 0.01 mN）和β - ar刺激后异丙肾上腺素（Delta分数缩短：72 +/- 5%对34 +/- 4%）的shCTR相比，AAV6-shB1产生的力更高。大规模基因表达分析显示，与未转导的EHTs相比，AAV6-shCTR仅显示少数差异调节基因（< 20），而AAV6-shB1诱导基因表达显著变化（bbb250基因），表明β (1)-AR敲低本身决定了结果。没有一个受调控的基因指出明显的脱靶效应来解释更高的力产生。此外，可以排除β (2)-AR信号的代偿性调节或突出的β (1)-AR下游靶点的变化。总之，我们在eht中有效敲除β (1)-AR后显示出矛盾的更高的力产生和异丙肾上腺素反应。我们的研究结果1)揭示了特异性β （1)-AR敲低后基因调控的复杂性，而不是通过转录干扰导致的非特异性失调，2)挑战了关于心脏β (1)-AR作用的经典假设，3）可能为体内b-AR功能丧失研究开辟了新的途径。
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.