背景:伪酶,活性酶的催化缺陷变体,具有广泛的调节功能。ADP-核糖水解酶样1(ADPRHL1),一种假酶,属于一小群ADP-核糖水解酶,缺乏催化活性所必需的氨基酸残基,根据越来越多的证据,可能在心脏发育中起重要作用。然而,ADPRHL1在此过程中的具体功能尚未阐明。探讨ADPRHL1在心脏中的作用,我们建立了第一个ADPRHL1基因敲除的体外人胚胎干细胞模型.
方法:使用CRISPR/Cas9系统,我们在人胚胎干细胞(hESC)H9系中产生了ADPRHL1敲除。使用化学上确定的和无异种的方法将细胞分化成心肌细胞。我们使用共聚焦激光显微镜检测钙瞬变和微电极阵列(MEA)来评估ADPRHL1缺乏心肌细胞的电生理活性。此外,我们通过BulkRNA测序和蛋白质印迹研究了ADPRHL1的细胞机制。
结果:结果表明,心肌细胞中ADPRHL1的缺失导致异常粘附,以及钙瞬变和电生理活动的扰动。我们还发现,这些心肌细胞中粘着斑形成的破坏是由于ROCK-肌球蛋白II途径的过度上调。值得注意的是,抑制ROCK和肌球蛋白II可有效恢复ADPRHL1缺陷型心肌细胞的局灶性粘附,并改善电传导和钙活性。
结论:我们的发现表明,ADPRHL1通过调节ROCK-肌球蛋白II途径在维持心肌细胞的正常功能中起关键作用,提示其可作为治疗ADPRHL1相关疾病的潜在药物靶点。
Pseudoenzymes, catalytically deficient variants of active enzymes, have a wide range of regulatory functions. ADP-ribosylhydrolase-like 1 (ADPRHL1), a pseudoenzyme belonging to a small group of ADP-ribosylhydrolase enzymes that lacks the amino acid residues necessary for catalytic activity, may have a significant role in heart development based on accumulating evidence. However, the specific function of ADPRHL1 in this process has not been elucidated. To investigate the role of ADPRHL1 in the heart, we generated the first in vitro human embryonic stem cell model with an ADPRHL1 knockout.
Using the CRISPR/Cas9 system, we generated ADPRHL1 knockout in the human embryonic stem cell (hESC) H9 line. The cells were differentiated into cardiomyocytes using a chemically defined and xeno-free method. We employed confocal laser microscopy to detect calcium transients and microelectrode array (MEA) to assess the electrophysiological activity of ADPRHL1 deficiency cardiomyocytes. Additionally, we investigated the cellular mechanism of ADPRHL1 by Bulk RNA sequencing and western blot.
The results indicate that the absence of ADPRHL1 in cardiomyocytes led to adhered abnormally, as well as perturbations in calcium transients and electrophysiological activity. We also revealed that disruption of focal adhesion formation in these cardiomyocytes was due to an excessive upregulation of the ROCK-myosin II pathway. Notably, inhibition of ROCK and myosin II effectively restores focal adhesions in ADPRHL1-deficient cardiomyocytes and improved electrical conduction and calcium activity.
Our findings demonstrate that ADPRHL1 plays a critical role in maintaining the proper function of cardiomyocytes by regulating the ROCK-myosin II pathway, suggesting that it may serve as a potential drug target for the treatment of ADPRHL1-related diseases.