■研究人骨髓间充质干细胞(hMSCs)中线粒体形态对细胞外基质硬度的机械响应以及AMP激活蛋白激酶(AMPK)在调节线粒体机械响应中的作用。
■两种聚丙烯酰胺(PAAm)水凝胶,杨氏模量为1kPa的软材料和刚度为20kPa的软材料,通过改变丙烯酰胺和双丙烯酰胺的单体浓度制备。然后,在柔软和坚硬的PAAm水凝胶上培养hMSCs,并使用激光共聚焦显微镜观察线粒体形态的变化。进行蛋白质印迹以确定AMPK的表达和激活。一种与线粒体稳态相关的蛋白质。此外,AMPK激活剂A-769662和抑制剂化合物C在柔软和坚硬的基质上调节AMPK的激活,分别,观察线粒体的形态变化。
■当凝胶硬度发生变化时,hMSC中线粒体的形态显示出异质性。在1kPa软基质上,74%的线粒体表现出致密,细长的丝状网络结构,而在20kPa刚性矩阵上,高达63.3%的线粒体呈碎片状或点状,分布稀疏。Western印迹结果表明,硬基质上磷酸化的AMPK(p-AMPK)/AMPK比率是软基质上的1.6倍。免疫荧光检测结果显示,p-AMPK在硬基质上的表达升高,并表现为核定位,表明细胞内AMPK的激活随着细胞外基质硬度的增加而持续增加。当软基质上的hMSCs用AMPK激活剂A-769662处理时,线粒体从丝状网络形态转变为碎片形态,丝状网络的比例从74%下降到9.5%。此外,用化合物C抑制AMPK促进了硬基质上的线粒体融合,并显著减少了点状线粒体的产生。
■细胞外基质硬度通过激活AMPK调节hMSCs线粒体形态。刚性基质促进AMPK激活,导致线粒体裂变和随后的线粒体碎片化。基质硬度对线粒体形态的影响可以通过改变AMPK磷酸化水平来逆转。
UNASSIGNED: To investigate the mechanical responses of mitochondrial morphology to extracellular matrix stiffness in human mesenchymal stem cells (hMSCs) and the role of AMP-activated protein kinase (AMPK) in the regulation of mitochondrial mechanoresponses.
UNASSIGNED: Two polyacrylamide (PAAm) hydrogels, a soft one with a Young\'s modulus of 1 kPa and a stiff one of 20 kPa, were prepared by changing the monomer concentrations of acrylamide and bis-acrylamide. Then, hMSCs were cultured on the soft and stiff PAAm hydrogels and changes in mitochondrial morphology were observed using a laser confocal microscope. Western blot was performed to determine the expression and activation of AMPK, a protein associated with mitochondrial homeostasis. Furthermore, the activation of AMPK was regulated on the soft and stiff matrixes by AMPK activator A-769662 and the inhibitor Compound C, respectively, to observe the morphological changes of mitochondria.
UNASSIGNED: The morphology of the mitochondria in hMSCs showed heterogeneity when there was a change in gel stiffness. On the 1 kPa soft matrix, 74% mitochondria exhibited a dense, elongated filamentous network structure, while on the 20 kPa stiff matrix, up to 63.3% mitochondria were fragmented or punctate and were sparsely distributed. Western blot results revealed that the phosphorylated AMPK (p-AMPK)/AMPK ratio on the stiff matrix was 1.6 times as high as that on the soft one. Immunofluorescence assay results revealed that the expression of p-AMPK was elevated on the hard matrix and showed nuclear localization, which indicated that the activation of intracellular AMPK increased continuously along with the increase in extracellular matrix stiffness. When the hMSCs on the soft matrix were treated with A-769662, an AMPK activator, the mitochondria transitioned from a filamentous network morphology to a fragmented morphology, with the ratio of filamentous network decreasing from 74% to 9.5%. Additionally, AMPK inhibition with Compound C promoted mitochondrial fusion on the stiff matrix and significantly reduced the generation of punctate mitochondria.
UNASSIGNED: Extracellular matrix stiffness regulates mitochondrial morphology in hMSCs through the activation of AMPK. Stiff matrix promotes the AMPK activation, resulting in mitochondrial fission and the subsequent fragmentation of mitochondria. The impact of matrix stiffness on mitochondrial morphology can be reversed by altering the level of AMPK phosphorylation.