Cullin-RING泛素连接酶(CRL)调节心脏中的许多生物过程,并与调节心脏肥大有关。本研究旨在鉴定心肌细胞(CM)中新型的肥大调节CRLs。采用使用siRNA介导的耗竭和自动显微镜的功能基因组方法来筛选新生大鼠CM中的细胞大小调节CRL。通过3H-异亮氨酸掺入确认筛选命中。在筛选的43个目标中,siRNA介导的Fbxo6,Fbxo45和Fbxl14的消耗导致细胞大小减小,而在基础条件下,Fbxo9,Fbxo25,Fbxo30,Fbxo32,Fbxo33,Cullin1,Roc1,Ddb1,Fbxw4和Fbxw5的消耗导致细胞大小显着增加。在用去氧肾上腺素(PE)刺激的CM中,Fbxo6,Fbxo25,Fbxo33,Fbxo45和Fbxw4的消耗进一步增加了PE诱导的肥大。作为一个概念证明,通过横向主动脉缩窄术(TAC)分析CRLFbox25,导致与对照动物相比,Fbxo25蛋白浓度增加4.5倍.在细胞培养中,siRNA介导的Fbxo25耗竭导致CM细胞大小增加约37%,3H-异亮氨酸掺入增加约41%。消耗Fbxo25导致Anp和Bnp上调。总之,我们鉴定出13个新的CRLs为CM肥大的正或负调节因子.其中,CRLFbox25进一步表征,作为心脏肥大的潜在调节剂。
Cullin-RING ubiquitin ligases (CRL) regulate numerous biological processes in the heart and have been implicated in regulating cardiac hypertrophy. This study aimed to identify novel hypertrophy-modulating CRLs in cardiomyocytes (CM). A functional genomic approach using siRNA-mediated depletion and automated microscopy was employed to screen for cell size-modulating CRLs in neonatal rat CM. Screening hits were confirmed by 3H-isoleucine incorporation. Of 43 targets screened, siRNA-mediated depletion of Fbxo6, Fbxo45, and Fbxl14 resulted in decreased cell size, whereas depletion of Fbxo9, Fbxo25, Fbxo30, Fbxo32, Fbxo33, Cullin1, Roc1, Ddb1, Fbxw4, and Fbxw5 led to a markedly increased cell size under basal conditions. In CM stimulated with phenylephrine (PE), depletion of Fbxo6, Fbxo25, Fbxo33, Fbxo45, and Fbxw4 further augmented PE-induced hypertrophy. As a proof-of-concept, the CRLFbox25 was analysed by transverse aortic constriction (TAC) resulting in a 4.5-fold increase in Fbxo25 protein concentrations compared to control animals. In cell culture, siRNA-mediated depletion of Fbxo25 resulted in a ∼ 37% increase in CM cell size and ∼41% increase in 3H-isoleucine incorporation. Depleting Fbxo25 resulted in upregulation of Anp and Bnp. In summary, we identified 13 novel CRLs as positive or negative regulators of CM hypertrophy. Of these, CRLFbox25 was further characterized, as a potential modulator of cardiac hypertrophy.