Explore the roles of intercellular communication in cardiomyocyte proliferation and renewal.

摘要 心力衰竭（HF）是美国发病率和死亡率的主要原因，预计其患病率将上升 随着总人口的老龄化。心肌梗死（MI）、慢性心脏超负荷或瓣膜疾病 通常会导致心肌细胞（CM）的丢失。然而，成年人的内源性增殖能力有限， CM阻碍CM更新并有助于HF的发展。有趣的是，最近的研究表明， 缺氧促进CM增殖，并改善心肌损伤后的恢复，表明内源性 参与CM增殖的途径可以被激活，并且足以诱导CM更新，而不需要遗传学上的改变。 在健康和患病的心脏中进行操作。因此，促进内生CM更新是一个 治疗HF的有前景的治疗方法。然而，缺氧诱导CM的机制 扩散在很大程度上仍不为人所知。脯氨酰羟化酶结构域蛋白（PHD）被广泛认为是 氧气传感器PHDs是否参与缺氧诱导的CM增殖和更新尚不清楚。此外，本发明还 内皮细胞（EC）充当对环境线索（例如氧气和营养物）的“第一反应者”。是 尚不清楚心脏EC-CM通信是否在缺氧诱导的CM增殖中起作用。因此，我们认为， 探索内皮PHDs在CM增殖中的作用对于理解CM的基本机制至关重要。 机制参与内源性CM更新，并将为我们提供新的方法来治疗HF。 我们最近已经证明，PHD 2/3的EC特异性敲除（eKO）促进CM增殖， 改善心功能，预防MI引起的心室衰竭。从机制上讲，我们发现， 是相关蛋白（雅普），器官大小控制和CM增殖的关键球员，被特异性激活 在PHD 2/3 eKO小鼠的CM中。单细胞RNA测序（scRNA-seq）分析显示，apelin（Apln），一种 GPCR配体通过HIF-2a在PHD 2/3 eKO小鼠的心脏EC中显著上调。我们进一步 证明Apln有效地激活CM中的雅普并促进CM增殖。值得注意的是，HIF-1 α的缺失 2a或Apln消除了在PHD 2/3 eKO小鼠中观察到的对心脏功能的有益作用。更 重要是，在新生或成年阶段的小鼠中CM特异性缺失Apln受体（AplnR）抑制雅普 激活和心脏功能恶化。这些数据使我们假设内皮PHD介导的 旁分泌机制通过Apln/AplnR通路在CM增殖和更新中起关键作用。为了验证这一 假设，我们将阐明潜在的分子机制，内皮PHD介导的旁分泌 Hippo-YAP信号通路调节CM中的Hippo-YAP信号传导。此外，我们还将研究Apln/AplnR的重要作用， 在CM增殖和正常心脏功能中的作用。最后，我们将研究Apln/AplnR通路在细胞凋亡中的作用， 在心力衰竭的小鼠模型中。

ABSTRACT Heart failure (HF) is a leading cause of morbidity and mortality in the US and its prevalence is expected to rise with the aging of the general population. Myocardial infarction (MI), chronic cardiac overload or valvular diseases often lead to the loss of cardiomyocytes (CMs). However, the limited endogenous proliferation capacity of adult CM impedes CM renewal and contributes to the development of HF. Interestingly, recent studies indicate that hypoxia promotes CM proliferation and improves recovery after myocardial injury, suggesting that endogenous pathways involved in CM proliferation can be activated and are sufficient to induce CM renewal without genetic manipulation in both healthy and diseased hearts. Therefore, promotion of endogenous CM renewal is a promising therapeutic approach to treat HF. However, the mechanisms involved in hypoxia-induced CM proliferation remain largely unknown. Prolyl hydroxylase domain proteins (PHDs) are widely considered as the oxygen sensors. Whether PHDs are involved in hypoxia-induced CM proliferation and renewal is unknown. Further, endothelial cells (ECs) act as the “first-responder” to environmental cues such as oxygen and nutrients. It is unclear whether cardiac EC-CM communication plays a role in hypoxia-induced CM proliferation. Therefore, exploring the role of endothelial PHDs in CM proliferation is of critical importance for understanding the basic mechanisms involved in endogenous CM renewal and will provide us novel approaches to treat HF. We have recently demonstrated that EC-specific knockout (eKO) of PHD2/3 promoted CM proliferation, improved cardiac function, and prevented ventricular failure induced by MI. Mechanistically, we discovered that yes-associated protein (YAP), a key player of organ size control and CM proliferation, was specifically activated in CMs of PHD2/3 eKO mice. Single-cell RNA sequencing (scRNA-seq) analysis revealed that apelin (Apln), a GPCR ligand, was markedly upregulated in cardiac ECs of PHD2/3 eKO mice via HIF-2a. We further demonstrated that Apln potently activated YAP in CMs and promoted CM proliferation. Notably, deletion of HIF- 2a or Apln in ECs eliminated the beneficial effects on cardiac function observed in PHD2/3 eKO mice. More importantly, CM-specific deletion of Apln receptor (AplnR) in mice at neonatal or adult stages inhibited YAP activation and deteriorated heart function. These data lead us to hypothesize that an endothelial PHD-mediated paracrine mechanism plays a key role in CM proliferation and renewal via Apln/AplnR pathway. To test this hypothesis, we will elucidate the underlying molecular mechanism by which endothelial PHD-mediated paracrine pathways regulate Hippo-YAP signaling in CMs. In addition, we will study the essential role of Apln/AplnR pathway in CM proliferation and normal heart function. Last, we will investigate the role of Apln/AplnR pathway in mouse models of heart failure.