A proteomic approach to characterize the human cardiomyocyte response to Doxorubicin exposure
Introduction
While the Anthracycline Doxorubicin (DOX) is a widely used and effective chemotherapeutic agent, it can lead to cardiotoxicity, and ultimately heart failure (HF) in some patients. HF is a complex disease with both a genetic and environmental component making risk prediction challenging. To gain insight into the mechanistic basis of DOX-induced cardiotoxicity and its contribution to HF, we investigated global protein expression changes in response to a pharmacologically-relevant dose of DOX in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).
Methods
iPSC-CMs were generated from three healthy individuals and treated with either a sub-lethal dose of DOX (0.5 uM) or a water vehicle control (VEH) for 24 hours. We replicated the treatment three times in one of the individuals. The 10 samples were processed using label-free mass spectrometry, wherein the proteins were first extracted, digested into peptides, separated by liquid chromatography, and then analyzed based on their mass-to-charge ratio to determine their relative abundances across different conditions. We used data from the technical replicates to remove unwanted technical variation in the data. Protein abundances between DOX- and VEH-treated iPSC-CMs were used to test for differentially expressed (DE) proteins using a linear mixed effects model with DOX treatment modeled as a fixed effect and Individual as a random effect.
Results
We detected expression for 3,384 proteins across samples including cardiac-specific proteins such as TNNT2, MYH7 and ACTN2. Samples are highly correlated within a treatment group, and DOX treatment accounts for most of the variation in the data. We identified 223 DE proteins (adj.P.Val < 0.05) between DOX and VEH treatment groups. The majority of DE proteins are downregulated in response to DOX treatment (82% of proteins; logFC < 0 ) including ANKR1, RSL1D1, CARD19, TRIM63, POLR2E, and DDX27 proteins. DE proteins are enriched for biological processes related to cytoplasmic translation, ribosomal biogenesis, amide biosynthesis, RNA processing, and p53 signal transduction compared to all expressed proteins. Consistent with the involvement of post-transcriptional and stress response processes following DOX treatment, DE proteins are enriched in cellular compartments including ribosomes, the nucleolus, spliceosomes and stress granules. To test whether the proteins that are DE due to DOX treatment are involved in susceptibility to HF, we collated 41 genes in loci previously associated with HF and restricted the set to those that are expressed in our data (9 genes). We find two of these gene products to be DE in response to DOX in our data (22%) including the transcriptional regulators GTF2I and SPEN.
Conclusion
Our data thus demonstrate that exposing human cardiomyocytes to a clinically-relevant concentration of DOX induces hundreds of protein expression changes, including proteins encoded by genes in loci associated with HF. Our model may therefore capture some of the contributors to DOX-induced cardiotoxicity and HF.