TEAD-independent mechanisms of YAP function in cardiomyocytes

Adult mammalian hearts exhibit limited regenerative capacity due to the restricted renewal of cardiomyocytes. Recent studies reveal that mammalian hearts exhibit transient regenerative potential within a short time frame after birth, suggesting a regulatory mechanism that prevents adult hearts from initiating a regenerative response to cardiac injury. Here, we discovered that an active form of YAP, named YAP6SA, which is not inhibited by the Hippo signaling pathway and does not interact with TEADs, induces cardiomyocyte cell cycle re-entry. In addition, YAP6SA interacts with scaffold protein MPDZ to regulate Rho GTPases and promote cell cycle progression in cardiomyocytes (CMs). Importantly, YAP6SA overexpression is well tolerated in mammalian hearts. These findings provide new insights into YAP function in cardiomyocytes. Overall design: Wildtype ICR mice were subcutaneously injected with 1*10^11 viral genomes of AAV9 virus containing cTnT-GFP, cTnT-YAP5SA, or cTnT-YAP6SA expression constructs at P6. Two hearts from each virus were collected at P9 and cardiomyocyte nuclei were enriched using anti-PCM1 pulldown. RNA was isolated using the RNeasy Plus Micro kit and poly-A enriched, non-directional libraries were generated by Novogene America and sequenced on the Illumina NovaSeq6000. Reads were mapped to the mouse genome (mm10, Ensembl GRCm38.94) using STAR. Lowly expressed genes were removed within each condition using median log2-transformed counts per gene per million mapped reads of 1, and a union set of genes was generated from each condition. Differential expression analysis was performed using edgeR using a general linear model framework with an additional covariate to mitigrate an apparent batch effect.

成年哺乳动物心脏因心肌细胞（cardiomyocytes）更新受限，再生能力极为有限。近期研究表明，哺乳动物心脏在出生后短期内具备短暂的再生潜能，这提示存在一种调控机制，可阻止成年心脏对心脏损伤启动再生应答。
本研究发现，一种不被Hippo信号通路（Hippo signaling pathway）抑制、且不与TEAD转录因子（TEADs）结合的Yes相关蛋白（YAP）活性变体YAP6SA，可诱导心肌细胞重新进入细胞周期。此外，YAP6SA可与支架蛋白MPDZ结合，调控Rho GTP酶（Rho GTPases）并促进心肌细胞（CMs）的细胞周期进程。值得注意的是，在哺乳动物心脏中过表达YAP6SA具有良好的耐受性。上述研究结果为阐明YAP在心肌细胞中的功能提供了全新视角。
整体实验设计：
于出生后第6天（P6）对野生型ICR品系小鼠皮下注射1×10^11病毒基因组的腺相关病毒9型（AAV9），其携带cTnT-GFP、cTnT-YAP5SA或cTnT-YAP6SA表达构建体；于出生后第9天（P9）收集各组2颗心脏，通过抗PCM1抗体磁珠下拉富集心肌细胞核。使用RNeasy Plus Micro试剂盒（RNeasy Plus Micro kit）提取RNA，随后由诺禾致源美国分部（Novogene America）构建polyA富集的非定向文库，并在Illumina NovaSeq6000测序平台上完成测序。使用STAR软件将测序reads比对至小鼠基因组（mm10，Ensembl GRCm38.94）。对每个实验组，以每百万比对reads中基因的中位数log2转换计数≥1作为阈值，过滤低表达基因，并整合各组的基因集合得到总基因集。采用edgeR软件，基于广义线性模型框架，并引入额外协变量以缓解明显的批次效应，进行差异表达基因分析。