Department of Clinical Pharmacology and Precision Medicine, William Harvey Research Institute, Barts and The London Faculty of Medicine and Dentistry, Queen Mary University of London, London, UK.
Building NIHBC 10 - Clinical Center, Bethesda, Maryland, USA.
J Biol Chem. 2024 Jul;300(7):107470. doi: 10.1016/j.jbc.2024.107470. Epub 2024 Jun 13.
Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function, but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function. We developed a murine model in which it was possible to conditionally delete ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Deletion of ric-8b led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis, and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodeling in response to cardiac ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of ric-8b led to loss of activation of the L-type calcium channel through the β-adrenergic pathways. Using fluorescence resonance energy transfer-based assays, we showed ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gα) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gα gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.
对胆碱酯酶抑制剂（Ric-8 蛋白）的耐药性参与调节 G 蛋白功能，但它们在心脏中的潜在生理重要性知之甚少。在本研究中，我们评估了对胆碱酯酶抑制剂 8b（Ric-8b）耐药性在确定心脏收缩功能中的作用。我们开发了一种小鼠模型，在成年动物中添加他莫昔芬后，可以在心脏组织中条件性删除 ric-8b。Ric-8b 的缺失导致在给予他莫昔芬几天后使用超声心动图测量的收缩性严重降低。心室组织的组织学分析显示肌细胞大小高度可变、明显纤维化和细胞凋亡增加。RNA 测序显示，Ric-8b 缺失导致心脏转录重塑，涉及细胞外基质和炎症。磷酸蛋白质组学分析显示与肌球蛋白轻链 2 相关的磷酸肽明显下调。在细胞水平上，Ric-8b 的缺失导致通过β肾上腺素能途径激活 L 型钙通道的丧失。通过使用荧光共振能量转移基于测定，我们表明 Ric-8b 蛋白选择性地与刺激 G 蛋白、Gαs 相互作用。我们探讨了是否使用类似的方法在小鼠中删除心脏组织中的 Gnas（编码 Gα 的基因）会导致类似的表型。心室中 Gα 基因的条件性缺失对收缩功能和心脏组织学产生了类似的影响。我们得出结论，Ric-8b 对于维持心脏收缩功能至关重要，可能通过与刺激 G 蛋白的相互作用以及肌球蛋白轻链 2 的下游磷酸化来实现。
