| Objective:The achievements of antineoplastic drug discovery have increased survival in patients with cancer,but treatment-induced cardiotoxicity is a leading noncancer-related cause of acute and late onset morbidity and mortality in cancer patients on antineoplastic drugs such as melphalan—increasing clinic case reports have documented that it could induce cardiotoxicity including severe arrhythmias and heart failure.As the mechanism by which melphalan impairs cardiac cells remains poorly understood,here we aimed to use cardiomyocytes derived from human induced pluripotent stem cells（hiPSC-CMs）to investigate the cellular and molecular mechanisms of melphalan-induced cardiotoxicity and to explore potential targeted therapeutics.Methods:First,human induced pluripotent stem cells were cultured and highly enriched hiPSC-CMs were generated using a small molecule-guided differentiation protocol and 3D cardiosphere formation.hiPSC-CMs were treated with clinically relevant doses of melphalan.To characterize the influence of melphalan on cell survival,cell viability and apoptosis were quantified by cell viability assays,detecting activated caspase-3/7,and quantitative real-time polymerase chain reaction（qRT-PCR）.Second,to acknowledge the effect of melphalan on cardiomyocyte function,ca2+transient assays were carried out and qRT-PCR was used to detect the expression of Ca2+channels and cardiomyocyte structure related genes.Third,global proteomics was conducted to investigate underlying mechanisms of melphalan induced cardiotoxicity in hiPSC-CMs.Finally,apply potential signaling inhibitors to treat hiPSC-CMs with melphalan and measure cell viability,contractility,and global transcriptomics to verify the role of the specific signaling pathway in melphalan-induced cardiotoxicity.Results:（1）Melphalan induced dose-and time-dependent cell death in hiPSC-CMs.（2）Melphalan treatment increased BAX expression and promoted early-stage apoptosis in hiPSC-CMs at a dose-dependent manner.（3）Melphalan altered Ca2+handling and the expression of Ca2+channels and cardiomyocyte structure related genes in hiPSC-CMs at a dose-dependent manner.（4）Global proteomic profile was significantly changed in melphalan-treated hiPSC-CMs.The dysregulated proteins mainly involved in oxidative stress,apoptotic process,cell adhesion,cardiovascular system development,heart contraction,p53 signaling pathway,et al.（5）Melphalan elevated intracellular and mitochondrial reactive oxygen species and increased the expression of oxidative stress related genes in hiPSC-CMs at a dose-dependent manner.（6）The antioxidant N-acetyl-L-cysteine（NAC）attenuated melphalan-induced cell death and oxidative stress.（7）Melphalan induced dysfunctional contractility in hiPSC-CMs,which could be attenuated by NAC supplementation.（8）Global transcriptomic profile was markedly altered in melphalan-treated hiPSC-CMs,which could be extraordinarily mitigated by NAC supplementation.The overlapping pathways that melphalan and NAC regulated included oxidative stress,extracellular matrix organization,cardiac muscle contraction,cell apoptosis,cytokine-cytokine interaction,p53 signaling pathway,TGF-βsignaling pathway,et al.Conclusions:（1）Melphalan had deleterious effects on hiPSC-CMs as indicated by significant cell death,early-stage apoptosis,excessive reactive oxygen species,deranged Ca2+handling,dysfunctional contractility,and altered global transcriptomic and proteomic profiles in a dose-dependent manner.（2）Melphalan induces cardiotoxicity through oxidative stress in hiPSC-CMs and several signaling pathways including the p53 and TGF-βsignaling pathways were implicated in melphalan-induced cardiotoxicity.（3）Antioxidant NAC could attenuate melphalan-induced cardiotoxicity.28 figures,22 tables,145 references... |
