Balbi Carolina, Milano Giuseppina, Fertig Tudor E, Lazzarini Edoardo, Bolis Sara, Taniyama Yoshiaki, Sanada Fumihiro, Di Silvestre Dario, Mauri Pierluigi, Gherghiceanu Mihaela, Lüscher Thomas F, Barile Lucio, Vassalli Giuseppe
Laboratory of Cellular and Molecular Cardiology, Istituto Cardiocentro Ticino, Lugano, Switzerland.
Center for Molecular Cardiology, University of Zurich, Zurich, Switzerland.
Theranostics. 2021 Mar 23;11(12):5634-5649. doi: 10.7150/thno.57243. eCollection 2021.
Although a small number of cardiomyocytes may reenter the cell cycle after injury, the adult mammalian heart is incapable of a robust cardiomyocyte proliferation. Periostin, a secreted extracellular matrix protein, has been implicated as a regulator of cardiomyocyte proliferation; however, this role remains controversial. Alternative splicing of the human periostin gene results in 6 isoforms lacking sequences between exons 17 and 21, in addition to full-length periostin. We previously showed that exosomes (Exo) secreted by human cardiac explant-derived progenitor cells (CPC) carried periostin. Here, we aimed to investigate their cell cycle activity. CPC were derived as the cellular outgrowth of cultured cardiac atrial explants. Exo were purified from CPC conditioned medium using size exclusion chromatography. Exosomal periostin was analyzed by Western blotting using a pair of antibodies (one raised against aa 537-836, and one raised against amino acids mapping at exon 17 of human periostin), by ELISA, and by cryo-EM with immune-gold labeling. Cell cycle activity was assessed in neonatal rat cardiomyocytes, in human induced pluripotent stem cell (iPS)-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. The role of periostin in cell cycle activity was investigated by transfecting donor CPC with a siRNA against this protein. Periostin expression in CPC-secreted exosomes was detected using the antibody raised against aa 537-836 of the human protein, but not with the exon 17-specific antibody, consistent with an isoform lacking exon 17. Periostin was visualized on vesicle surfaces by cryo-EM and immune-gold labeling. CPC-derived exosomes induced cell proliferation in neonatal rat cardiomyocytes both and , in human iPS-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. Exo promoted phosphorylation of focal adhesion kinase (FAK), actin polymerization, and nuclear translocation of Yes-associated protein (YAP) in cardiomyocytes. Knocking down of periostin or YAP, or blocking FAK phosphorylation with PF-573228 nullified Exo-induced proliferation. A truncated human periostin peptide (aa 22-669), but not recombinant human full-length periostin, mimicked the pro-proliferative activity of exosomes. Our results show, for the first time, that CPC-secreted exosomes promote cardiomyocyte cell cycle-reentry via a short periostin isoform expressed on their surfaces, whereas recombinant full-length periostin does not. These findings highlight isoform-specific roles of periostin in cardiomyocyte proliferation.
虽然少数心肌细胞在损伤后可能重新进入细胞周期，但成年哺乳动物心脏的心肌细胞无法进行强劲的增殖。骨膜蛋白是一种分泌型细胞外基质蛋白，被认为是心肌细胞增殖的调节因子；然而，这一作用仍存在争议。人类骨膜蛋白基因的可变剪接除了产生全长骨膜蛋白外，还产生6种在第17和21外显子之间缺乏序列的异构体。我们之前发现，人心脏外植体来源的祖细胞（CPC）分泌的外泌体（Exo）携带骨膜蛋白。在此，我们旨在研究它们的细胞周期活性。CPC是从培养的心脏心房外植体的细胞生长物中获得的。使用尺寸排阻色谱法从CPC条件培养基中纯化Exo。通过使用一对抗体（一种针对第537 - 836氨基酸残基，另一种针对人骨膜蛋白第17外显子定位的氨基酸）进行蛋白质印迹分析、酶联免疫吸附测定（ELISA）以及冷冻电镜免疫金标记来分析外泌体中的骨膜蛋白。在新生大鼠心肌细胞、人诱导多能干细胞（iPS）来源的心肌细胞以及成年大鼠心肌梗死后的心肌细胞中评估细胞周期活性。通过用针对该蛋白的小干扰RNA（siRNA）转染供体CPC来研究骨膜蛋白在细胞周期活性中的作用。使用针对人骨膜蛋白第537 - 836氨基酸残基的抗体检测到CPC分泌的外泌体中骨膜蛋白的表达，但未使用第17外显子特异性抗体检测到，这与缺乏第17外显子的异构体一致。通过冷冻电镜和免疫金标记在囊泡表面观察到骨膜蛋白。CPC来源的外泌体在新生大鼠心肌细胞、人iPS来源的心肌细胞以及成年大鼠心肌梗死后的心肌细胞中均诱导细胞增殖。Exo促进心肌细胞中粘着斑激酶（FAK）的磷酸化、肌动蛋白聚合以及Yes相关蛋白（YAP）的核转位。敲低骨膜蛋白或YAP，或用PF - 573228阻断FAK磷酸化可消除Exo诱导的增殖。一种截短的人骨膜蛋白肽（第22 - 669氨基酸残基），而非重组人全长骨膜蛋白，模拟了外泌体的促增殖活性。我们的结果首次表明，CPC分泌的外泌体通过其表面表达的短骨膜蛋白异构体促进心肌细胞重新进入细胞周期，而重组全长骨膜蛋白则无此作用。这些发现突出了骨膜蛋白异构体在心肌细胞增殖中的特异性作用。
