过表达GS1增强心肌球干细胞在缺血心肌中的存活和治疗作用

Cardiaosphere-derived Cell (CDC) therapy has shed unprecedented light on ischemic heart disease treatment due to its strong cardiac differentiation potential.However, the massive dead of transplanted CDC in ischemic myocardium limit their cardiac repair greatly. It is well-known that stem cells negotiate their surrounding nutritional and signaling environments and respond accordingly in functions and fates. Under ischemic environment, oxygen-independent glycolysis becomes the critical means for cells to generate ATP. To maintain this anaerobic metabolism for long-term survival of stem cell after severe and continuous ischemia, persistent glucose supply is required. Glycogen is a polymer of glucose residues in cytoplasm. Glycogen plays an important role in glucose metabolism and forms an energy reserve that can be quickly mobilized to meet the sudden need for energy. Recently we found glycogen synthase 1(GS1), a rate-limiting enzyme catalyze glycogen production, is a key regulator in the re-balance of metabolic dysfunction during ischemia. Because of its potential role in stem cell protect against ischemia, we hypothesis that overexpression of GS1 may contribute to the metabolic homeostasis of CDC in ischemic microenvironment and further enhance its cardiac repair and regeneration to ischemic myocardium. To test our hypothesis, GS1 overexpression adenovirus and mouse acute myocardial infarction model will be used. We will verify the regulation of GS1-overexpression on CDC glycogen metabolism and ATP content in vitro. Besides, further studies will demonstrate the effects of GS1 overexpression on CDC survival and therapeutic efficacy in vivo MI model. Furthermore, this study will elucidate the regulation mechanism of GS1 overexpression on CDC therapy upon glycogen metabolism, apoptosis and autophagy pathways. This study is dedicated to determine whether and how GS1 and it mediated glycogen metabolism enhances CDC survival and therapy under ischemia and provide new insight for the clinical application of endogenous cardiac stem cell-CDC.

心肌球干细胞(CDC)以其优良的心肌分化潜能已成为当前缺血性心脏病细胞治疗领域的研究热点，然而，细胞移植后大量的生理或病理性丢失是制约其移植疗效的关键瓶颈，从代谢调控角度能否实现CDC的内源性保护以增强其移植后的生存与移植疗效，尚不明确。我们前期研究发现，糖原合成酶1（GS1）是干细胞在缺血环境下改善供能障碍的关键调控分子，由此，GS1的过表达有望通过改善缺血CDC的供能障碍以增强其移植效能。本项目拟利用GS1过表达腺病毒感染CDC，借助体外缺血模型处理细胞，观察过表达GS1对CDC的保护效应；并采用小鼠急性心肌梗死模型，借助活体成像与组织学技术确认过表达GS1-CDC移植后的存活效率与移植疗效；进一步通过特异地阻断糖原合成与细胞自噬过程观察关键信号分子的改变，从而阐明GS1过表达介导的内源保护机制。本研究旨在从代谢调控角度探索保护心肌球干细胞的新策略，为其临床转化提供新依据。

目前，临床上对于心肌梗死后的纤维化重构尚无有效干预手段，尽管伴随着争议，但细胞移植仍然是一种极具潜力的心肌修复策略。心肌球细胞(Cardiosphere-derived Cells, CDC)是一种心肌间质细胞，因可于悬浮培养中形成心肌球而得名，具有成血管分化等部分干细胞特性，其治疗潜力已在近年多个临床研究中得以证实(Lancet, 2012; J Am Coll Cardiol, 2014; Circ Res, 2017 and Circ Res, 2018)。细胞移植后大量的生理或病理性丢失是制约细胞疗法发展的关键瓶颈，本研究中我们以CDC为研究对象，通过体内外研究，探讨通过改善移植细胞糖原代谢进而增强移植疗效的科学假设。在研究中，我们发现：（1）过表达糖原合成酶1（GS1）可以显著提升CDC中的糖原含量，胞内糖原储存可以帮助CDC对抗体外缺氧缺血环境，显著减少缺氧缺血诱导的细胞死亡；（2）在小鼠心梗模型中，我们移植了过表达GS1的CDC与对照CDC，通过一系列的病理和心功能评估手段，结果显示过表达GS1对CDC的体内治疗作用仅有部分提升。过表达GS1尽管显著提高CDC在小鼠梗死心肌中的存活率，但对CDC的促心肌修复功能并无显著影响，该组实验鼠的心功能、梗死面积、细胞坏死及微血管密度均无显著差异。（3）在机制研究层面，我们证实在体外缺氧缺血环境中自噬参与了过表达GS1介导的细胞保护。但由于过表达GS1的CDC并未在体内表现出优于对照CDC的心肌修复功能，所以我们进一步去探讨造成这种现象的原因和机制。我们发现，经数次传代扩增后，过表达GS1的CDC会更快地出现衰老样表型，表现为部分细胞增殖减慢且β-gal染色呈阳性，提示过表达GS1所致的糖原积累在作为能量储存的同时也可能产生应激作用促进细胞衰老，进而限制其体内移植疗效。本研究以GS1为抓手，从调控糖原代谢的角度探索保护心肌球干细胞的新策略，阐明了过表达GS1对CDC移植治疗梗死心肌的“双刃剑”作用，揭示了细胞移植前衰老的重要现象，为CDC的临床转化提供了新依据。