Global gene expression profiles of cellular response in human induced pluripotent stem cell-derived cardiomyocytes to melphalan treatment

Purpose: Evaluate the molecular changes associated with melphalan-induced cardiotoxicity and rescue by N-acetyl-L-cysteine (NAC) supplementation. Methods: RNA-Seq analysis was performed to analyze global transcriptome profiles of hiPSC-CMs treated with DMSO (Control group), 20 µM melphalan (Mel group), and 20 µM melphalan with 1 mM NAC (Mel+NAC group), respectively. The Illumina TruSeq technology was used to prepare RNA-Seq libraries, and next-generation sequencing was done via an Illumina HWI-ST1276. RNA sequence reads were aligned to the human reference genome (GRCh38) using STAR v2.5. HTSeq v0.6.1 was used to count the read numbers mapped of each gene, and then Fragments Per Kilobase Million (FPKM) was calculated to estimate gene abundance. Differential expression analysis was performed using the DESeq2 R package (2_1.6.3). The resulting P-values were adjusted using the Benjamini and Hochberg's approach. Results: As detected by RNA-Seq, more genes were down-regulated than up-regulated by the treatment of melphalan, whereas NAC supplementation resulted in more genes being up-regulated than down-regulated. Among the top 10 up-regulated genes by melphalan treatment, 4 were direct p53 effectors (CDKN1A, EDXR, TNFRSF10C, and GDF15). And among the top 10 down-regulated genes by melphalan treatment, 5 were correlated to cell adhesion (CDH13, CNTN1, SDK1, CTNND2, and PARD3B). In addition, the up- and down-regulation of genes involved in oxidative stress (e.g., DUOX2 and NOX4) following melphalan treatment (Mel vs. control) was attenuated with NAC supplementation (Mel+NAC vs. Control). Similarly, the up- and down-regulation of genes involved in cardiac muscle contraction (e.g., Ca2+ handling proteins CACNA1C, RYR2 and CASQ2 and cardiac contractile proteins TNNC1, ACTC1, TNNC1) and cardiac conduction (e.g., ATP2B2 and ABCC9) following melphalan treatment (Mel vs. Control) was attenuated with NAC supplementation (Mel+NAC vs. Control). Conclusion: NAC ameliorates melphalan-induced alteration of hiPSC-CM transcriptomic profiles. RNA-Seq analysis was performed to compare global gene expression profiles of hiPSC-CMs treated with DMSO, 20 µM melphalan, and 20 µM melphalan with 1 mM NAC, respectively, in triplicate.

研究目的：评估美法仑（melphalan）诱导的心肌毒性相关分子改变，以及N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine, NAC）补充干预的挽救效应。
研究方法：分别对经二甲基亚砜（DMSO，对照组）、20 μM美法仑（美法仑组）、20 μM美法仑联合1 mM N-乙酰-L-半胱氨酸（美法仑+NAC组）处理的人诱导多能干细胞源性心肌细胞（human induced pluripotent stem cell-derived cardiomyocytes, hiPSC-CMs）开展RNA测序（RNA-Seq）分析，以解析其全局转录组谱。采用Illumina TruSeq技术构建RNA-Seq文库，通过Illumina HWI-ST1276平台完成下一代测序。使用STAR v2.5软件将测序读段比对至人类参考基因组（GRCh38）；采用HTSeq v0.6.1统计各基因的比对读段数，并通过每百万片段每千碱基（Fragments Per Kilobase Million, FPKM）计算基因丰度。差异表达分析使用DESeq2 R软件包（版本2_1.6.3）完成，所得P值通过Benjamini-Hochberg法进行校正。
研究结果：经RNA-Seq检测发现，美法仑处理组的下调基因数量多于上调基因；而NAC补充干预后，上调基因数量多于下调基因。美法仑处理上调的前10位基因中，有4个为p53直接效应靶基因（CDKN1A、EDXR、TNFRSF10C及GDF15）。美法仑处理下调的前10位基因中，有5个与细胞黏附功能相关（CDH13、CNTN1、SDK1、CTNND2及PARD3B）。此外，美法仑处理组（美法仑组 vs 对照组）中涉及氧化应激的基因（如DUOX2与NOX4）的表达变化，在NAC补充干预组（美法仑+NAC组 vs 对照组）中得到显著缓解。同样，美法仑处理组中涉及心肌收缩的基因（如钙离子转运蛋白CACNA1C、RYR2、CASQ2，以及心肌收缩蛋白TNNC1、ACTC1、TNNC1）和心脏传导相关基因（如ATP2B2与ABCC9）的表达异常，在NAC补充干预后也得到了有效缓解。
研究结论：NAC可改善美法仑诱导的人诱导多能干细胞源性心肌细胞转录组谱改变。本研究通过RNA-Seq分析，对分别经DMSO、20 μM美法仑、20 μM美法仑联合1 mM NAC处理的hiPSC-CMs进行了三次生物学重复，以比较其全局基因表达谱。