背景:我们先前证明了一个意大利家庭患有严重的扩张型心肌病(DCM),在年轻时就有猝死史,在编码LaminA/C蛋白(LMNA)的截短变体的Lmna基因中携带突变,R321X.当在异源系统中表达时,这种变体积累到内质网(ER),诱导未折叠蛋白反应(UPR)的PERK-CHOP途径的激活,ER功能障碍和凋亡率增加。这项工作的目的是分析靶向UPR是否可用于逆转与HL-1心脏细胞中LMNAR321X表达相关的ER功能障碍。
方法:使用稳定表达LMNAR321X的HL-1心肌细胞评估3种不同药物靶向UPR的能力,salubrinal,guanabenz和empagliflozin挽救ER压力和功能障碍。在这些细胞中,分析UPR和促凋亡途径的活化状态,监测磷酸-PERK的表达水平,磷酸-eIF2α,ATF4、CHOP和PARP-CL。此外,我们测量了ER依赖性细胞内Ca2+动力学作为适当ER功能的指标。
结果:我们发现salubrinal和guanabenz增加磷酸-eIF2α的表达水平,并下调LMNAR321X心肌细胞中的凋亡标志物CHOP和PARP-CL,维持所谓的适应性普遍定期审议。这些药物还恢复了ER处理这些心肌细胞中Ca2+的能力。有趣的是,我们发现,依帕列净下调凋亡标志物CHOP和PARP-CL,通过抑制LMNAR321X-心肌细胞中的PERK磷酸化,从而关闭UPR.此外,在依帕列净治疗后,ER稳态,在ER方面,在这些心肌细胞中也恢复了储存和释放细胞内Ca2+的能力。
结论:我们提供的证据表明,不同的药物,尽管干扰了普遍定期审议的不同步骤,能够抵消R321XLMNA心肌细胞的促凋亡过程并保持ER稳态。值得注意的是,两种测试药物,guanabenz和empagliflozin,已经在临床实践中使用,从而为受LMNAR321X相关心肌细胞影响的患者的即用治疗提供了临床前证据.
We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells.
HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal,
guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality.
We found that salubrinal and
guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes.
We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs,
guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes.