Mechanobiology Institute, National University of Singapore, Singapore, Singapore.
Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.
Cell Mol Biol Lett. 2023 May 11;28(1):39. doi: 10.1186/s11658-023-00446-9.
Tissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury.
We express optogenetic YAP (optoYAP) in three different cell lines. We characterised the behaviour and function of optoYAP using fluorescence imaging and quantitative real-time PCR of downstream YAP target genes. Mutant constructs were generated using site-directed mutagenesis. Nuclear localised optoYAP was functionally tested using wound healing assay.
Utilising optoYAP, which enables precise control of pathway activation, we show that YAP induces the expression of downstream genes involved in proliferation and migration. optoYAP can increase the speed of wound healing in H9c2 cardiomyoblasts. Interestingly, this is not driven by an increase in proliferation, but by collective cell migration. We subsequently dissect specific phosphorylation sites in YAP to identify the molecular driver of accelerated healing.
This study shows that optogenetic YAP is functional in H9c2 cardiomyoblasts and its controlled activation can potentially enhance wound healing in a range of conditions.
组织在受伤后需要再生以恢复功能。然而，这种再生能力在不同器官和物种之间存在显著差异。例如，在心脏中，一些物种在其整个生命周期中都保持着完全的再生能力，而人类心肌细胞修复损伤的能力有限。心肌梗死后，心肌细胞的功能受损，降低了心脏的泵血能力，导致心力衰竭。因此，需要在心肌梗死后恢复受损心脏的功能。我们在细胞培养中研究了 Yes 相关蛋白（YAP）的作用，YAP 是一种在生长中起关键作用的转录共调节因子，它在驱动损伤后修复中发挥作用。
我们在三种不同的细胞系中表达光遗传学 YAP（optoYAP）。我们使用荧光成像和下游 YAP 靶基因的定量实时 PCR 来描述 optoYAP 的行为和功能。使用定点突变生成突变构建体。使用伤口愈合测定法测试核定位的 optoYAP 的功能。
利用能够精确控制途径激活的 optoYAP，我们表明 YAP 诱导参与增殖和迁移的下游基因的表达。optoYAP 可以增加 H9c2 心肌细胞的伤口愈合速度。有趣的是，这不是由增殖增加驱动的，而是由细胞集体迁移驱动的。随后，我们对 YAP 中的特定磷酸化位点进行细分，以确定加速愈合的分子驱动因素。
这项研究表明，光遗传学 YAP 在 H9c2 心肌细胞中是功能性的，其受控激活可能在多种情况下增强伤口愈合。
