Molecular Cardiology Laboratory, Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria 3004, Australia.
J Mol Cell Cardiol. 2013 Jan;54:19-24. doi: 10.1016/j.yjmcc.2012.10.014. Epub 2012 Nov 5.
Autophagy is a process that removes damaged proteins and organelles and is of particular importance in terminally differentiated cells such as cardiomyocytes, where it has primarily a protective role. We investigated the involvement of inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) and its receptors in autophagic responses in neonatal rat ventricular myocytes (NRVM). Treatment with the IP(3)-receptor (IP(3)-R) antagonist 2-aminoethoxydiphenyl borate (2-APB) at 5 or 20 μmol/L resulted in an increase in autophagosome content, defined as puncta labeled by antibody to microtubule associated light chain 3 (LC3). 2-APB also increased autophagic flux, indicated by heightened LC3II accumulation, which was further enhanced by bafilomycin (10nmol/L). Expression of Ins(1,4,5)P(3) 5-phosphatase (IP(3)-5-Pase) to deplete Ins(1,4,5)P(3) also increased LC3-labeled puncta and LC3II content, suggesting that Ins(1,4,5)P(3) inhibits autophagy. The IP(3)-R can act as an inhibitory scaffold sequestering the autophagic effector, beclin-1 to its ligand binding domain (LBD). Expression of GFP-IP(3)-R-LBD inhibited autophagic signaling and furthermore, beclin-1 co-immunoprecipitated with the IP(3)-R-LBD. A mutant GFP-IP(3)-R-LBD with reduced ability to bind Ins(1,4,5)P(3) bound beclin-1 and inhibited autophagy similarly to the wild type sequence. These data provide evidence that Ins(1,4,5)P(3) and IP(3)-R act as inhibitors of autophagic responses in cardiomyocytes. By suppressing autophagy, IP(3)-R may contribute to cardiac pathology.
自噬是一种清除受损蛋白质和细胞器的过程,在终末分化细胞(如心肌细胞)中尤为重要,在这些细胞中,它主要具有保护作用。我们研究了肌醇(1,4,5)三磷酸(Ins(1,4,5)P(3))及其受体在新生大鼠心室肌细胞(NRVM)自噬反应中的作用。用 IP(3)受体(IP(3)-R)拮抗剂 2-氨基乙氧基二苯硼酸盐(2-APB)在 5 或 20μmol/L 浓度下处理会导致自噬体含量增加,这定义为微管相关轻链 3(LC3)抗体标记的斑点。2-APB 还增加了自噬流,表现为 LC3II 的积累增加,而巴弗洛霉素(10nmol/L)进一步增强了这一作用。表达 Ins(1,4,5)P(3)5-磷酸酶(IP(3)-5-Pase)以耗尽 Ins(1,4,5)P(3)也增加了 LC3 标记的斑点和 LC3II 含量,表明 Ins(1,4,5)P(3)抑制自噬。IP(3)-R 可以作为一种抑制性支架,将自噬效应物,beclin-1 与其配体结合域(LBD)隔离。GFP-IP(3)-R-LBD 的表达抑制了自噬信号,此外,beclin-1 与 IP(3)-R-LBD 共免疫沉淀。具有降低与 Ins(1,4,5)P(3)结合能力的 GFP-IP(3)-R-LBD 突变体与野生型序列相似,结合 beclin-1 并抑制自噬。这些数据提供了证据表明 Ins(1,4,5)P(3)和 IP(3)-R 作为心肌细胞自噬反应的抑制剂。通过抑制自噬,IP(3)-R 可能有助于心脏病理学。