新型内源性心肌保护分子Betaglycan分子调控网络及其外源融合肽干预研究

Our preliminary data have shown that high abundance of betaglycan is expressed in the cardiomyocytes. The present proposal　 was designed to determine the expression changes of betaglycan during cardiac ischemia reperfusion injury (IRI) and to identify its endogenous cardiac protective roles. We hypothesized that extracellular soluble betaglycan (e-betaglycan) as an isoform-selective TGF-β1 neutralizing agent exerts inhibitory effect on TGF-β signaling; and the intracellular domain of betaglycan (c-betaglycan) interacts with β-arrestin2 and GIPC to affect downstream molecules including p-BAD, TRL4, NF-κB and ERK by which betaglycan exhibits anti-apoptotic effects and attenuates Cx43, IK1, Ito channel remodeling during IRI. Moreover, two classic approaches including cardiac ischemic preconditioning and administration of stating drug were used to investigate whether betaglycan expression is up-regulated in both classic cardiac protective models and to identify the possibility of betaglycan to be a potential target for the prevention and treatment of heart disease. Finally, by in vivo administration of exogenous s-betaglycan or fused peptide of c-betaglycan respectively in both preventive and therapeutic intervention manner for IRI model, we are able to compare the efficacy between them in anti-IRI process and improving cardiac function. The present proposal will firstly demonstrate betaglycan is a critical regulator in anti-apoptotic process and anti-electrical remodeling in cardiomyocytes under ischemia condition. These findings will certainly expand our understanding of endogenous cardiac protectors. Discovery of endogenous betaglycan and corresponding exogenous fused peptide will unravel a novel cardioprotective strategy and prove a potential therapeutic approach.

本课题在首次证实Betaglycan（BG）在心肌细胞存在高丰度表达基础上，揭示其在心肌缺血再灌注损伤(IRI)模型中的变化规律，阐明其作为内源性心肌保护分子分别通过胞外片段（e-BG）对TGF-β信号产生负性调控和胞内片段（c-BG）与β-arrestin2，GIPC分子共耦联对下游p-BAD，TRL4, NF-κB和ERK等信号分子产生调节，从而影响心肌细胞凋亡和Cx43，IK1，Ito等离子通道重构发挥心肌保护作用；采用缺血预适应以及他汀药物两种经典心肌保护方法确定BG 是否成为防治IRI的干预靶点；最终通过在体实验给予其外源性e-BG或胞浆融合肽c-BG 进行预防和治疗性干预研究，分别比较二者抗IRI的效力和对心功的影响。本研究将首次证明BG是对IRI过程中细胞凋亡和电重构调控的关键分子，拓展我们对内源性心肌保护分子的认识，并为心力衰竭和心律失常等重大心脏疾病提供新策略和干预手段。

本课题在首次证实转化生长因子III型受体(TGFBR3，Betaglycan）在心肌细胞存在高丰度表达基础上，揭示其在心肌重构中的作用及其机制。我们发现：①辛伐他汀上调TGFBR3是其产生保护心脏作用的新机制。辛伐他汀抑制了由心肌梗死引起的心肌纤维化进程，同时上调了TGFBR3的表达并降低ERK1/2 和 JNK的磷酸化。在乳鼠心肌成纤维细胞中，辛伐他汀可上调TGFBR3蛋白水平，并且敲除TGFBR3可逆转辛伐他汀对细胞增殖和胶原生成的抑制作用。同时，辛伐他汀可抑制由血清引起的ERK1/2和JNK信号活性增强，敲除TGFBR3可逆转此效应。在成纤维细胞中TGFBR3通过与GIPC偶联抑制ERK1/2和JNK信号。②TGFBR3为心肌细胞凋亡新调控分子。发现在心肌梗死模型（MI）以及H2O2诱导的心肌细胞凋亡模型中TGFBR3蛋白表达呈现先升高后降低的变化。TGFBR3 促进H2O2对心肌细胞的凋亡作用，并激活p38MAPK信号通路。而敲除TGFBR3，呈现相反效应。过表达TGFBR3的转基因鼠（CS-TGFBR3 Tg）梗死面积显著增加且细胞凋亡率亦显著升高，同时，p38蛋白的磷酸化水平也随之上调；而在体内敲减TGFBR3则表现出相反的作用。TGFBR3通过激活p38信号通路促进由心肌缺血引起的心肌细胞凋亡，而抑制TGFBR3的表达可降低细胞凋亡并缓解心肌损伤，证明TGFBR3介导的p38信号调控了心肌细胞凋亡的发生。 ③TGFBR3调控心肌肥厚。在人心肌肥厚临床样本、异丙肾上腺素（ISO）诱导或胸主动脉结扎（TAC）诱导的小鼠心肌肥厚模型中TGFBR3 蛋白表达均显著升高。，ISO 和血管紧张素II（AngII）都可引起乳鼠心肌细胞TGFBR3 的表达增加。过表达TGFBR3导致 心肌细胞表面积增大，肥厚相关基因表达增加；而心肌细胞特异性敲减TGFBR3 则可预防ISO 和AngII 诱导的细胞肥大。发现CS-TGFBR3出现心肌肥厚的表型。采用连接AAV9 病毒TGFBR3shRNA 敲减小鼠心脏TGFBR3 表达，可预防ISO 和TAC 诱导的小鼠心肌肥厚。TGFBR3 通过与β-arrestin2 的相互作用，进而通过β-arrestin2 与CaMKII 相互作用激活CaMKII是TGFBR3 参与心肌肥厚发生的重要机制。