The pseudoenzyme ADPRHL1 affects cardiac function by regulating the ROCK pathway

Pseudoenzymes, catalytically deficient variants of active enzymes, have a wide range of regulatory functions. ADP-ribosylhydrolase like-1 (ADPRHL1), which belongs to a small group of ADP-ribosylhydrolase enzymes, lacks the amino acid residues required for catalytic activity, and is therefore considered a pseudoenzyme. Here, to investigate the role of ADPRHL1 in the heart, we established, for the first time, an in vitro ADPRHL1 knockout human myocardial cell culture model. These ADPRHL1 knockout cardiomyocytes adhered abnormally. However, the lack of ADPRHL1 disrupted the formation of focal adhesions in these cardiomyocytes by excessively upregulating the ROCK–myosin II pathway. Inhibitors of ROCK and myosin II effectively restored the focal adhesions in these ADPRHL1-deficient cardiomyocytes and improved electrical conduction and calcium activity. Our findings reveal that ADPRHL1 plays a major role in cardiac function by regulating the ROCK pathway, suggesting that it may serve as a potential drug target for the treatment of ADPRHL1-related diseases. To elucidate how ADPRHL1 deficiency prevents the formation of FAs, WT-CMs and KO-CMs were collected for global transcriptome analysis within 3 days after re-seeding.

假酶（Pseudoenzyme）是活性酶的催化缺陷型变体，具备广泛的调控功能。ADP-核糖基水解酶样蛋白1（ADP-ribosylhydrolase like-1, ADPRHL1）属于ADP-核糖基水解酶家族的一个小分支，其缺失催化活性所需的氨基酸残基，因此被认定为假酶。本研究为探究ADPRHL1在心脏中的生物学功能，首次构建了体外ADPRHL1敲除人心肌细胞培养模型。该模型中的ADPRHL1敲除心肌细胞表现出黏附异常。进一步研究发现，ADPRHL1缺失会通过过度上调ROCK-肌球蛋白II通路，破坏心肌细胞黏着斑（focal adhesions）的形成。ROCK与肌球蛋白II抑制剂可有效恢复ADPRHL1缺陷心肌细胞的黏着斑结构，并改善其电传导与钙活动。本研究结果揭示，ADPRHL1通过调控ROCK通路在心脏功能中发挥核心作用，提示其有望成为ADPRHL1相关疾病的潜在药物靶点。为阐明ADPRHL1缺失如何抑制黏着斑形成，我们在细胞重接种后的3天内收集野生型心肌细胞（WT-CMs）与敲除型心肌细胞（KO-CMs），进行全转录组分析。