From the Interdisciplinary Stem Cell Institute (K.E.H., A.R.W., M.A.B., W.Y., J.M.H.), University of Miami, Miller School of Medicine, FL.
Department of Cell Biology (K.E.H.), University of Miami, Miller School of Medicine, FL.
Circ Res. 2019 Apr 12;124(8):1184-1197. doi: 10.1161/CIRCRESAHA.118.314063.
Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 (retinoblastoma) and CDKN2a (cyclin-dependent kinase inhibitor 2a) are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear.
We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes, and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle reentry.
Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes revealed that RB1 and CDKN2a are upregulated at the onset of cardiac precursor specification, simultaneously with GATA4 (GATA-binding protein 4) homeobox genes PBX1 (pre-B-cell leukemia transcription factor 1) and MEIS1 (myeloid ecotropic viral integration site 1 homolog), and remain so until terminal cardiomyocyte differentiation. In both GATA4 hPSC cardiac precursors and postmitotic hPSC-cardiomyocytes, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the cardiac precursors stage, but not in terminally differentiated hPSC-cardiomyocytes, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in postmitotic hPSC-cardiomyocytes can be induced with transient, dual RB1, and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4 cells correlates with the degree of scar size reduction and endogenous cardiomyocyte mitosis, particularly in response to combined transendocardial injection of adult human hMSCs (bone marrow-derived mesenchymal stromal cells) and cKit cardiac cells.
Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4 cells and cardiomyocytes mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications.
虽然有报道称在成年哺乳动物心脏中存在罕见的心肌发生，但这种现象是源于心肌细胞的分化还是增殖仍存在争议。肿瘤抑制基因 RB1（视网膜母细胞瘤）和 CDKN2a（细胞周期蛋白依赖性激酶抑制剂 2a）是细胞周期调控的关键因子，但它们在人类心肌发生中的作用尚不清楚。
我们假设 RB1 和 CDKN2a 的发育性激活协同作用，导致人类心肌前体细胞（CPCs）的永久性细胞周期退出，从而推动其向成熟心肌细胞的终末分化，并且双重失活这些肿瘤抑制基因可促进心肌细胞的细胞周期再进入。
定向分化人类多能干细胞（hPSCs）为心肌细胞显示，RB1 和 CDKN2a 在心脏前体细胞特化开始时上调，与 GATA4（GATA 结合蛋白 4）同源盒基因 PBX1（前 B 细胞白血病转录因子 1）和 MEIS1（髓系成瘤病毒整合位点 1 同源物）同时上调，并一直持续到终末心肌细胞分化。在 GATA4 hPSC 心肌前体细胞和有丝分裂后 hPSC-心肌细胞中，RB1 均被高度磷酸化而失活。在 hPSC 分化过程中，RB1 的短暂、阶段特异性耗竭可增强心脏前体细胞阶段的心肌发生，但在终末分化的 hPSC-心肌细胞中则不然，这是通过细胞周期调控途径短暂地上调 GATA4 表达实现的，该途径涉及 CDKN2a。重要的是，在有丝分裂后 hPSC-心肌细胞中，短暂的双重 RB1 和 CDKN2a 沉默可诱导胞质分裂。在心肌细胞治疗后 MI 的猪模型中发现了该途径的相关性，提示该途径在体内的相关性，即在成年 GATA4 细胞中双重 RB1 和 CDKN2a 失活与疤痕大小减小和内源性心肌细胞有丝分裂的程度相关，尤其是在经心内膜注射成年人类 hMSCs（骨髓源性间充质基质细胞）和 cKit 心脏细胞后。
综上所述，这些发现揭示了 RB1 和 CDKN2a 在调控人类心肌发生过程中的细胞周期进展和分化中具有重要的协调作用。此外，在成年 GATA4 细胞和心肌细胞中短暂的双重 RB1 和 CDKN2a 失活至少部分介导了基于细胞的治疗在大型哺乳动物 MI 后模型中的有益作用，这一发现具有潜在的临床意义。
