ZNFX1调节NAD+与钙离子稳态抗心脏衰老作用机制研究

The incidence of cardiovascular disease increases linearly with age. Clearing the key regulatory molecules and drug targets of aging hearts is the bottleneck of research. We performed transcript sequencing, and found that lncRNA ZFAS1 was significantly up-regulated in aging mouse cardiac tissue. The expression level of ZNFX1 was significantly down-regulated. Overexpression of ZNFX1 in aging mice showed improved cardiac function and anti-aging effects on the heart. KEGG analysis demonstrated that overexpression of ZNFX1 significantly influenced the niacin and nicotinamide metabolism-related pathways and calcium ion pathways. Based on the above, we have established the following scientific hypothesis: (1) During the process of heart aging, the methylation of ZNFX1 promoter is increased, which causes the transcription of ZNFX1 to be terminated, and then the expression of antisense-lncRNA ZFAS1 is up-regulated by the "transcriptional competition" mechanism, further inhibiting SERCA2a, inducing calcium overload and causes aging of cardiomyocytes. (2) At the same time, ZNFX1 acts as a transcription factor to regulate the expression of NMNAT3 and results in a decrease in NMNAT3 and a limited synthesis of NAD+, which in turn leads to energy metabolism disorders in cardiomyocytes, which in turn leads to aging. Our research revealed new mechanism of cardiac aging, and provided effective drug targets for anti-aging.

心血管疾病的发生随年龄增长呈线性增加，明晰衰老心脏关键调节分子和药物靶点是研究的瓶颈问题。我们对衰老小鼠进行转录本测序，发现lncRNA ZFAS1在衰老小鼠心脏组织中高表达，而其正义编码基因ZNFX1表达水平显著下调。对衰老小鼠过表达ZNFX1后，小鼠心功和心脏衰老得到明显改善，KEGG分析显示，ZNFX1过表达后，烟酸和烟酰胺代谢相关通路及钙离子通路发生最明显改变。基于以上内容我们建立如下科研假设：（1）在心脏衰老过程中，ZNFX1启动子甲基化增加，使得ZNFX1转录终止，继而通过“转录竞争”机制促进ZFAS1表达上调，进一步抑制SERCA2a，诱发钙超载，导致心肌细胞衰老。（2）ZNFX1作为转录因子调节NMNAT3的表达，衰老中，ZNFX1表达下调使得NMNAT3减少，NAD+合成受限，继而导致心肌细胞能量代谢障碍，诱发衰老。本研究揭示了心脏衰老新的发病机制，提供了可行药物靶点。

衰老是多种重大慢性致死性疾病的主要诱因。研究发现，心血管病的发生随年龄增长呈线性增加，延缓衰老以及伴随的心功恶化是目前研究的重点，明晰衰老心脏关键调节分子和药物靶点是研究的瓶颈问题。我们对衰老小鼠心脏组织进行了转录本测序，通过差异基因筛选发现，lncRNA ZFAS1（ZNFX1 antisense RNA 1）在衰老小鼠心脏组织中表达显著上调。ZFAS1定位于20号染色体，由ZNFX1（zinc finger NFX1-type containing 1）基因的反义链转录而来，二者转录起始位点相近，ZNFX1启动子区域与ZFAS1编码区“头对头”相互交叠。我们发现ZNFX1在衰老小鼠心脏中表达水平显著下调，表明ZNFX1可能在衰老心脏中发挥重要的作用。本研究旨在探究ZNFX1对心脏衰老的调控作用及机制，为缓解心脏衰老功能障碍提供候选方案。年轻小鼠尾静脉注射腺相关病毒AAV9-shZNFX1敲低小鼠ZNFX1，相比于正常小鼠，敲低ZNFX1后小鼠出现心脏衰老表型，即心脏肥大、心脏舒张功能障碍。与年轻小鼠相比，年老小鼠中ZNFX1的表达水平显著降低，尾静脉注射AAV9-ZNFX1恢复衰老小鼠ZNFX1表达水平，能够抑制衰老相关蛋白表达，改善衰老心脏的舒张功能，延缓心脏衰老。在原代心肌细胞中，敲除ZNFX1显著诱导心肌细胞衰老表型，过表达ZNFX1则能够显著抑制D-gal诱导的细胞衰老。TMT相对定量蛋白组学检测和分析发现，过表达ZNFX1后衰老小鼠差异表达蛋白显著富集于呼吸链复合体和线粒体呼吸链细胞组分。差异表达蛋白ISD11和ACP同为铁硫蛋白的组分，引起了我们的关注。小鼠过表达ZNFX1能够显著恢复衰老心脏中ISD11和ACP的表达。本研究首次提出以ZNFX1表达含量为指标评估心脏老化程度，明确ZNFX1对衰老心脏功能障碍的调节作用，可为老年心血管病的治疗提供新的策略，也为预防心脏衰老提供有效的治疗靶点。