The Study On The Effect And Mechanism Of Doxorubicin-induced Cardiotoxicity Based On Transcriptomics And Metabolomics Analysis In Vitro

Objective: Doxorubicin belongs to anthracycline antibiotics with a strong and widespectrum antitumor activity,which is limited to clinical application due to the serious cardiotoxicity.The mechanism of doxorubicin induced cardiomyopathy is still not completely clear nowadays.Omics analysis can help us to systematically understand the process of the occurrence and development of doxorubicin induced cardiomyopathy.Methods: In the first part of the study,an RNA sequencing assay was performed in H9c2 cardiomyocytes samples with or without doxorubicin exposure,data was integrated with a microarray gene dataset titled GSE42177 downloaded from GEO database and analyzed using GEO2 R.The integrated DEGs was used to run GO and KEGG analysis with David database.PPI network analysis was performed using String database and visualized with Cytoscape software.LC-MS/MS assay was performed to detect the change of the metabolites in the supernatant samples of H9c2 cardiomyocytes.CCK-8 assay together with the annexin-V/PI double staining assay were performed to discover the apoptosis level.ChIP-seq and ChIP-q PCR were used to detect the enrichment level of H3K9 ac in the transcription initiation region of Slc19a2.In the second part,we built a conditioned medium model between H9c2 cardiomyocytes and cardiac fibroblasts for investigating doxorubicin induced cardiac fibrosis.CCK-8 assay and Ed U assay were used for detecting the proliferation level of cardiac fibroblasts.The expression of the fibrotic markers including Col1a1,Col3a1,Tgfb1 and α-SMA were determined by q PCR,western blotting and immunofluorescence staining representing the activation level in cardiac fibroblasts.Quasi-targeted metabolomics and LC-MS were used for identification and quantification of the potential factors in the supernatant of H9c2 cardiomyocytes.The effect of HNMT/NMH axis was detected by loss of function and rescue assay.Results: A total of 51 integrated DEGs were identified,24 of which were upregulated.6 of the upregulated DEGs including the specific thiamine transporter titled Slc19a2(upregulated to about 4.42-fold and 2.62-fold compared to the control group in RNAseq and GSE42177 separately)were enriched in the GO clustering term titled integral component of plasma membrane.LC-MS/MS analysis revealed that the level of thiamine was decreased to about 78% in the supernatant samples of H9c2 cardiomyocytes after doxorubicin exposure.Additional thiamine ranged from 1 to8mg/L rescued doxorubicin induced apoptosis.All these results indicated that the upregulated thiamine transport was an endogenous mechanism counteracting doxorubicin induced cardiotoxicity in H9c2 cardiomyocytes.Results of ChIP-seq and ChIP-q PCR indicated that the enrichment of H3K9 ac in the transcriptome initiated region was upregulated after doxorubicin exposure,which can be regulated by sh RNA targeting Slc19a2 or thiamine supplement.The proliferation and activation levels were significantly upregulated after doxorubicin exposure in the conditioned medium model.Quasi-targeted metabolomics analysis suggested that NMH might be a key factor in the supernatant H9c2 cardiomyocytes,which was quantified by LC-MS and was subsequently verified by NMH supplement with a concentration of 0.15ng/m L to the conditioned medium from the control group.For mechanism research,we were focusing on HNMT by the metabolism pathway analysis.Western blotting and q PCR results revealed that HNMT was upregulated after doxorubicin exposure.Knockdown of Hnmt attenuated doxorubicin induced NMH release in H9c2 cardiomyocytes and the elevation of proliferation and activation levels in cardiac fibroblasts,which were rescued by NMH supplement,indicating that the HNMT/NMH axis was involved in doxorubicin induced fibrosis in our conditioned medium model.Conclusion: Thiamine transport was involved in counteracting doxorubicin induced apoptosis via a H3K9 acetylation related feedback loop in H9c2 cells.Conditioned medium of doxorubicin induced H9c2 cardiomyocytes promoted the proliferation and activation of cardiac fibroblasts via HNMT/NMH axis.