CREG抑制心肌细胞自噬死亡和纤维化发生的溶酶体调控机制研究

Cardiac fibrosis is a common feature of end-stage heart diseases, independently of their etiologies, and the leading cause of morbidity and mortality in our country. Loss of cardiomyocytes due to apoptosis, necrosis, and/or autophagic-associated cell death is a key initiating mechanism of cardiac fibrosis. Autophagy is an intracellular lysosomal degradation process operating in homeostatic clearance of organelles and protein aggregates. However, pressure overload and stress elicit a robust autophagic response and lead to the death of cardiomyocytes, although the underlying mechanisms remain poorly understood. Cellular Repressor of E1A-stimulated Genes (CREG) is an evolutionarily conserved glycoprotein that promotes cell differentiation and homeostasis in response to stress and damage. Our preliminary studies demonstrated that CREG is highly expressing in embryonic and adult myocardium. Moreover, reduced CREG expression in CREG heterozygous (CREG+/-) mice enhances cardiac fibrosis during aging or after angiotensin II treatment, correlating with increased autophagic activities in cardiomyocytes. Base on these findings, we hypothesize that suppression of CREG expression might contribute to cardiomyocyte death and cardiac fibrosis by activating the autophagic-lysosomal pathway. To test this hypothesis, we propose the following aims: 1) To explore the relationship of reduced CREG expression and autophagic associated cardiomyocyte death and cardiac fibrosis using CREG+/- mice; 2) CREG loss of function analysis will be performed in primary culture cardiomyocytes to determine the mechanisms that CREG regulates autophagic-associated cardiomyocyte death; 3)Using cardiomyocyte- specific CREG transgenic mice to investigate the effect of CREG overexpression on angiotensin-induced autophagic activation, cardiomyocyte injury and cardiac fibrosis. Completion of the proposed experiments will provide new insights into the mechanisms whereby CREG antagonizes cardiomyocyte death and cardiac fibrosis by modulating the autophagic-lysosomal activity. It may also be implicated in the development of new therapeutic approaches.

心肌纤维化是多种心脏疾病恶性演进的共同病理基础，对抗或逆转纤维化的发生是有效降低心脏疾病死亡率的重要研究方向。各种病理因素诱发的心肌细胞死亡是纤维化发生的始动机制。近期研究证实，过度自噬激活是心肌细胞死亡的直接病理基础，但其发生机制尚未阐明。本室前期研究发现，CREG是成熟心肌细胞中高表达的溶酶体稳态调控因子，其表达减少引起心肌细胞自噬激活和心肌纤维化发生。本研究尝试从心肌细胞内稳态调控入手，通过CREG低表达的杂合子小鼠和原代心肌细胞两种模型，阐明CREG在心肌自噬死亡和纤维化发生中的作用及机制；并通过心肌特异性过表达CREG的转基因小鼠对抗心肌纤维化的治疗研究，揭示CREG调控心肌细胞自噬-溶酶体稳态、保护心肌存活、对抗纤维化发生的作用机制。期望本研究的顺利完成，能深入揭示以CREG为代表的自噬-溶酶体调控分子在心肌细胞损伤和纤维化发生中的作用，为心脏疾病防治提供新思路。

各种病理刺激诱发的心肌细胞死亡是纤维化的始动因素，但其机制尚未阐明。研究表明，CREG是维持心脏稳态的重要因子，也是调控溶酶体功能的关键蛋白，但其在心肌纤维化中的作用仍不清楚。因此，本研究旨在明确CREG在病理性心肌损伤及心肌纤维化中的作用，并阐明其机制。研究内容主要包括3个方面：（1）明确CREG表达减少与心肌自噬功能障碍、心肌细胞损伤和心肌纤维化的相关关系。（2）阐明CREG调控心肌细胞损伤和心肌纤维化的自噬溶酶体机制。（3）明确CREG是否可作为心肌纤维化的干预靶点。我们采用血管紧张素II（AngII）诱导的心肌纤维化模型和心肌缺血再灌注（MIR）损伤两种模型进行探讨。首先，在AngII诱导的心肌纤维化模型中：AngII处理后心肌中CREG蛋白表达明显降低，且心脏收缩功能减低及心肌纤维化程度随CREG蛋白水平降低而加重，表明CREG可能参与这一病理过程。进一步研究发现，CREG降低后心肌细胞中自噬功能障碍，表现为自噬流受阻。应用外源性重组CREG蛋白，可明显改善自噬功能障碍，并挽救AngII刺激引起的心肌损伤。应用自噬抑制剂氯喹（CQ）后，CREG蛋白的挽救作用消失。体外细胞学实验表明，CREG通过与小G蛋白Rab相互作用，调控自噬的终末环节，即自噬溶酶体的形成。第二，在MIR模型中：MIR损伤后心肌中CREG蛋白表达显著降低，CREG蛋白水平与MIR后心功能呈正相关，而与MIR心肌细胞凋亡呈负相关。MIR后，CREG表达减低的心肌组织表现为自噬功能障碍，即自噬底物发生堆积；而给予外源性重组CREG蛋白后自噬被激活。应用自噬抑制剂CQ后，明确了CREG蛋白的保护作用来自于对自噬的改善。细胞学研究表明，CREG能通过增强溶酶体的发生及功能从而激活自噬，对抗心肌细胞凋亡。上述结果表明，CREG在不同病理情况下的心肌损伤及心肌纤维化中发挥重要作用，其机制是通过调控自噬的终末环节，即自噬溶酶体形成来发挥作用。本项目明确了CREG是参与心肌纤维化发生的重要因子，并阐明了其自噬溶酶体机制，拓展了对心肌纤维化机制的理解，同时为其治疗提供了新的靶点。