β-肾上腺素受体通过PI3K信号通路调控心肌细胞的增殖

Conventionally, it is thought that adult cardiomyocytes cannot regenerate because of the loss of proliferation capacity. A recent study demonstrated that surgical resection of the ventricular apex in 1-day-old mice stimulated a regenerative response that appeared to restore the damaged heart to its normal anatomy and function, although the molecular mechanisms underlying this process remain unclear. It's been shown that PI3K activities are highly regulated during cardiac development, high in fetal and neonatal periods but low in the adult, which is approximately parallel to cardiomyocytes proliferation transition. We have established βAR-PI3K crosstalk in adult mouse heart. Taken together, we hypothesize that tonic inhibition or activation of βAR changes the activities of PI3K signaling pathway, and consequently regulates cardiomyocytes proliferation in neonatal mammalian heart. The preliminary experiments showed that βAR blockade significantly reduced DNA synthesis of cardiomyocytes in newborn mice, with cardiac PI3K activity promptly decreased. In the current project, we seek to elucidate the regulation of βAR on cardiomyocyte proliferation using a neonatal mouse model. Also, we will explore the role of PI3K signaling pathway in the signal transduction of this regulation and its key downstream effector(s). This in vivo study may yield novel insights for understanding the signaling mechanism that responsible for the cardiomyocyte cell cycle regulation，which would be most beneficial for studies directed towards myocardial regeneration and repair following cardiac injury.

传统认为，成熟心肌细胞由于失去增殖能力而无法再生。最近有实验证明，切除新生小鼠心尖，可刺激受损心脏再生，并恢复其形态和功能，但调控这一过程的分子机制尚不清楚。研究表明，在心脏发育过程中PI3K活性的变化与心肌细胞增殖能力的转变密切相关，而在成年小鼠心脏中βAR与PI3K信号通路之间存在交互作用(crosstalk)。因此，我们提出如下科学假说：阻断/激活βAR，可通过改变PI3K信号通路的活性而调控心肌细胞的增殖。初步实验结果提示，在新生小鼠，阻断βAR使心脏中PI3K的活性明显下降，并显著抑制心肌细胞中DNA的合成。本课题拟以新生小鼠心脏为模型，通过体内实验研究βAR在心肌细胞增殖能力转变过程中的调控作用，并探索PI3K信号通路在这一调控中的意义，进而筛选调控心肌细胞增殖的关键信号蛋白，为心肌细胞周期调控的信号转导机制提供新的见解，为促进心肌损伤后的再生和修复提供新的思路。

项目背景：在发育早期的心脏中，虽然可检测到β肾上腺素受体（βAR）及其配体，甚至配体合成所需的酶，但却没有兴奋-收缩偶联。在成年小鼠中，慢性βAR活化会引起心肌的肥大。我们之前的数据表明PI3K的活性在心肌发育过程中受到高度调控，在胚胎和新生阶段该激酶活性较高，而在成年阶段活性则较低。我们还发现，改变β2AR可引起心肌特异性PI3K信号通路的相应变化，说明在心脏中存在βAR和PI3K间的交互作用。本项目的主要目的是澄清该作用的生理效应，尤其是对心肌增殖的调控，并探讨可能参与这种调控的信号途径。.研究内容：新生C57BL/6小鼠先经腹腔注射非选择性βAR阻滞剂普萘洛尔（10mg/kg），一小时后再注射EdU（5乙炔-2'-脱氧尿苷，50mg/kg），4小时后取心脏组织制备石蜡切片，通过免疫荧光染色检测EdU和α-辅肌动蛋白-2以观察心肌细胞的增殖情况。为检测相关信号激酶，新生小鼠用普萘洛尔处理30分钟，随后裂解心脏组织，检测裂解液中的PI3K活性及Akt、GSK-3α/β、FoxOs、mTOR、p70S6K、S6和ERK1/2等蛋白的磷酸化水平。.主要结果：阻断βAR显著降低新生小鼠心脏中处于增殖状态的心肌细胞数（处理组19.0±3.1/视野 vs 对照组30.4±2.2/视野，p<0.05）和增殖指数（增殖心肌细胞数/总细胞核数，处理组6.2±1.0% vs 对照组11.7±0.9%，p<0.05）。用普萘诺尔阻断新生小鼠的βAR 引起心脏中PI3K的活性显著下降，并导致PI3K下游多个效应因子的磷酸化水平降低，包括Akt、GSK-3α/β、FoxO1/3a。但急性阻断βAR对于mTOR/p70S6K/S6信号通路的活性不引起功能性改变，对于ERK1/2的活化也没有影响。.科学意义：本研究不但证实了在新生小鼠心脏中存在βAR-PI3K交互作用，还阐明了此通路在心肌细胞增殖中的重要作用。深入研究这一信号转导活动将有助于理解围生期心脏发育的机理，特别是心肌细胞由增殖向肥大转换的调控机制，并可能为治疗急性心梗和心衰提供新的治疗途径。