Hippo signaling pathway maintains sinoatrial node homeostasis

The sinoatrial node (SAN) functions as pacemaker of the heart to initiate and drive rhythmic heartbeats. The Hippo signaling pathway is a fundamental pathway for heart development and regeneration. Although abnormalities of Hippo pathway are associated with cardiac arrhythmias in human patients, yet its role in the SAN is unknown. We found that Lats1/2 inactivation caused severe sinoatrial node dysfunction (SND; sick sinus syndrome). Compared to the controls, Lats1/2 CKO mutants exhibited dysregulated calcium handling and increased fibrosis in the sinoatrial node, indicating Lats1/2 function through both cell-autonomous and non-cell-autonomous mechanisms. Notably, the Lats1/2 CKO phenotype was rescued by genetic deletion of Yap and Taz in the CCS, and these rescued mice had normal sinus rhythm and reduced fibrosis of the sinoatrial node, indicating that Lats1/2 function through Yap and Taz. CUT&Tag sequencing data showed that Yap regulates genes critical for calcium homeostasis such as Ryr2 and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as Tgf-ß1 and Tgf-ß3. Consistently, Lats1/2 CKO mutants had decreased Ryr2 expression and increased Tgf-ß1 and Tgf-ß3 expression compared with control mice. We reveal for the first time that the canonical Hippo-Yap pathway has a pivotal role in functional homeostasis of the sinoatrial node. Overall design: We investigated key regulators of the Hippo pathway in the sinoatrial node by conditionally inactivating the Hippo signaling kinases Lats1 and Lats2 using the tamoxifen-inducible, cardiac conduction system (CCS)-specific Cre driver Hcn4CreERT2 and Lats1 and Lats2 conditional knockout (CKO) alleles. In addition, the Hippo signaling effectors Yap and Taz were conditionally inactivated in the sinoatrial node. To determine the function of the Hippo signaling pathway in the sinoatrial node and other CCS components, we conducted a series of physiological and molecular experiments including telemetry ECG recording, echocardiography, Masson's Trichrome staining, calcium imaging, immunostaining, Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing using antibodies against Yap1 or H3K4me3, qRT-PCR, and Western blotting. R56HL142704 and R01HL142704 to Jun Wang; T32HL07676 to Rich Gang Li; R01HL163277, R01HL136389, and R01HL147108 to Na Li; American Heart Association SDG19840000 and TPA970606 to Jun Wang; American Heart Association 902940 to Mingjie Zheng; American Heart Association 903251 to Rich Gang Li; American Heart Association EIA936111 to Na Li.

窦房结（sinoatrial node, SAN）是心脏的起搏位点，负责启动并驱动节律性心跳。Hippo信号通路是调控心脏发育与再生的核心通路。尽管人类患者体内Hippo通路异常与心律失常存在关联，但其在窦房结中的功能仍未明确。
我们的研究发现，Lats1/2失活会引发严重的窦房结功能障碍（sinoatrial node dysfunction, SND；即病窦综合征）。与对照组相比，Lats1/2条件性敲除（conditional knockout, CKO）突变体的窦房结中出现钙处理失调与纤维化程度升高的现象，表明Lats1/2通过细胞自主性与非细胞自主性两种机制发挥调控作用。
值得注意的是，在心脏传导系统（cardiac conduction system, CCS）中遗传敲除Yap与Taz可挽救Lats1/2 CKO的表型，挽救后的小鼠可维持正常窦性心律，且窦房结纤维化程度显著降低，这证实Lats1/2通过Yap和Taz介导其功能。
切割靶标与标签测序（Cleavage Under Targets and Tagmentation, CUT&Tag）数据显示，Yap可调控钙稳态关键基因如Ryr2，以及编码参与细胞间通信与纤维化诱导的旁分泌因子的基因如转化生长因子-β1（TGF-β1）与转化生长因子-β3（TGF-β3）。与之相符的是，与对照组小鼠相比，Lats1/2 CKO突变体中Ryr2表达水平下调，而TGF-β1与TGF-β3的表达水平上调。
本研究首次揭示经典Hippo-Yap通路在窦房结功能稳态中发挥关键调控作用。

整体实验设计：我们利用他莫昔芬诱导型心脏传导系统特异性Cre驱动工具Hcn4CreERT2与Lats1、Lats2条件性敲除等位基因，对窦房结内Hippo通路的关键调控因子展开研究。此外，我们还在窦房结中条件性失活了Hippo通路效应因子Yap与Taz。为明确Hippo通路在窦房结及其他心脏传导系统组分中的功能，我们开展了一系列生理与分子实验，包括遥测心电图记录、超声心动图、Masson三色染色、钙成像、免疫染色、使用Yap1或H3K4me3抗体的CUT&Tag测序、实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）以及蛋白质印迹实验。

资助信息：本研究得到以下项目支持：Jun Wang的R56HL142704与R01HL142704；Rich Gang Li的T32HL07676；Na Li的R01HL163277、R01HL136389与R01HL147108；Jun Wang的美国心脏协会（American Heart Association）项目SDG19840000与TPA970606；Mingjie Zheng的美国心脏协会项目902940；Rich Gang Li的美国心脏协会项目903251；Na Li的美国心脏协会项目EIA936111。