Department of Pharmacology (The State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China.
Research Unit of Noninfectious Chronic Diseases in Frigid Zone, Chinese Academy of Medical Sciences, Harbin, China.
Clin Transl Med. 2022 Jun;12(6):e908. doi: 10.1002/ctm2.908.
The development of heart ageing is the main cause of chronic disability, disease and death in the elderly. Ample evidence has established a pivotal role for significantly reduced mitophagy in the ageing heart. However, the underlying mechanisms of mitophagy deficiency in ageing heart are little known. The present study aimed to explore the underlying mechanisms of lncRNA LOC105378097 (Senescence-Mitophagy Associated LncRNA, lncR-SMAL) actions on mitophagy in the setting of heart ageing.
The expression of lncR-SMAL was measured in serum from different ages of human and heart from different ages of mice through a quantitative real-time polymerase chain reaction. The effects of lncR-SMAL on heart function of mice were assessed by echocardiography and pressure-volume measurements system. Cardiac senescence was evaluated by hematoxylin-eosin staining, senescence-associated β-galactosidase staining, flow cytometry and western blot analysis of expression of ageing related markes p53 and p21. Cardiomyocyte mitophagy was assessed by western blot, mRFP-GFP-LC3 adenovirus particles transfection and mito-Keima staining. Interaction between lncR-SMAL and Parkin was validated through molecular docking, RNA immunoprecipitation (RIP) and RNA pull-down assay. Ubiquitination assay was performed to explore the molecular mechanism of Parkin inhibition. The effects of lncR-SMAL on mitochondrial function were investigated through electron microscopic examination, JC-1 staining and oxygen consumption rates analysis.
The heart-enriched lncR-SMAL reached the expression crest in the serum of human at an age of 60. Exogenously overexpression of lncRNA SMAL deteriorated cardiac function exactly as natural ageing and inhibited the associated cardiomyocytes mitophagy by depressing Parkin protein level. Improved heart ageing and mitophagy caused by Parkin overexpression were reversed by lncR-SMAL in mice. In contrast, the loss of lncR-SMAL in AC16 cells induced the upregulation of Parkin protein and ameliorated mitophagy and mitochondrial dysfunction, resulting in alleviated cardiac senescence. Besides, we found the interaction between lncR-SMAL and Parkin protein through computational docking analysis, pull-down and RIP assay. This would contribute to the promotive effect of lncR-SMAL on Parkin ubiquitination and decrease Parkin protein stability.
The present study for the first time demonstrates a heart-enriched lncRNA, SMAL, that inhibits the mitophagy of cardiomyocytes via the downregulation of Parkin protein, which further contributes to heart ageing and cardiac dysfunction in natural ageing mice.
心脏衰老的发展是老年人慢性残疾、疾病和死亡的主要原因。大量证据表明，线粒体自噬的显著减少在衰老心脏中起着关键作用。然而，衰老心脏中线粒体自噬缺失的潜在机制知之甚少。本研究旨在探讨长链非编码 RNA LOC105378097（衰老相关线粒体自噬长链非编码 RNA，lncR-SMAL）在心脏衰老过程中对线粒体自噬作用的潜在机制。
通过实时定量聚合酶链反应检测不同年龄人类血清和不同年龄小鼠心脏中的 lncR-SMAL 表达。通过超声心动图和压力-容积测量系统评估 lncR-SMAL 对小鼠心脏功能的影响。通过苏木精-伊红染色、衰老相关β-半乳糖苷酶染色、流式细胞术和衰老相关标志物 p53 和 p21 表达的 Western blot 分析评估心脏衰老。通过 Western blot、mRFP-GFP-LC3 腺病毒颗粒转染和 mito-Keima 染色评估心肌细胞线粒体自噬。通过分子对接、RNA 免疫沉淀（RIP）和 RNA 下拉实验验证 lncR-SMAL 与 Parkin 之间的相互作用。通过泛素化实验探讨 Parkin 抑制的分子机制。通过电子显微镜检查、JC-1 染色和耗氧率分析研究 lncR-SMAL 对线粒体功能的影响。
在人类血清中，心脏特异性丰富的 lncR-SMAL 在 60 岁时达到表达高峰。外源性过表达 lncRNA SMAL 可恶化心脏功能，与自然衰老完全一致，并通过抑制 Parkin 蛋白水平抑制相关的心肌细胞线粒体自噬。在小鼠中，Parkin 过表达改善的心脏衰老和线粒体自噬被 lncR-SMAL 逆转。相反，AC16 细胞中 lncR-SMAL 的缺失导致 Parkin 蛋白上调，并改善线粒体自噬和线粒体功能障碍，从而减轻心脏衰老。此外，我们通过计算对接分析、下拉和 RIP 实验发现 lncR-SMAL 与 Parkin 蛋白之间存在相互作用。这有助于促进 lncR-SMAL 对 Parkin 泛素化的作用，并降低 Parkin 蛋白的稳定性。
本研究首次证明了一种心脏特异性丰富的 lncRNA SMAL，通过下调 Parkin 蛋白抑制心肌细胞的线粒体自噬，进一步导致自然衰老小鼠的心脏衰老和心功能障碍。
