RBM20 promotes doxorubicin-induced cardiotoxicity by Atp6v0a1 alternative splicing

Abstract Background & Objective: Doxorubicin (dox), an effective chemotherapy drug, is extremely limited in clinical practice by its lethal cardiotoxicity. Whereas dysregulation of splice-regulatory networks has been reported in cardiomyopathies, the role of RNA binding motif protein 20 (RBM20), an adaptive splice regulator, in doxorubicin-induced cardiotoxicity (DIC) remained to be elucidated. Purpose Here we aimed to show the role of RBM20 in DIC and elucidate the relationship between alternative splicing and lysosome acidification impairment. Method Mice were treated with intraperitoneal injection of cumulative 20mg/kg doxorubicin with four equal injections over a period of 2 weeks to induce DIC. The contribution of RBM20 in cardiotoxicity was evaluated in conditional cardiomyocyte-specific RBM20 overexpression (RBM20tg/+/Myh6-CreERT) mice. The regulatory factor of RBM20 was determined through luciferase assay and chromatin immunoprecipitation in vitro. Autophagic flux was evaluated by transmission electron microscope in RBM20tg/+/Myh6-CreERT mice and by lysotracker and a tandem fluorescence RFP-GFP-LC3 reporter system in RBM20 overexpressed or knockdown cardiomyocyte. RBM20-dependent splicing events were identified by rMATS 3.2.5 on the RNA-sequencing data. Results In the DIC human myocardium and murine model, we observed significant elevation of RBM20 in cardiomyocytes. Cardiomyocyte-specific RBM20 overexpression resulted in severe cardiac dysfunction reflected by dilated ventricles, declined left ventricular ejection fraction, increased chamber stiffness and lower survival rate. After screening for potential upstream transcription factors, c-Jun was identified to bind and upregulate RBM20 directly. The inhibition of c-Jun by SR 11302 could partially mitigate DIC in vitro. Mechanistically, RBM20 was positively related with autophagic flux blockade and impaired lysosome acidification by shifting Atp6v0a1, the a1-subunit of the V0 domain of vacuolar H+-ATPases, toward an exon 6-7 exclusion isoform without altering total transcript levels. By restoring exon 6-7-containing ATP6V0a1 level, we were able to reduce DIC by restoring normal autophagic flux. Conclusion Overall, our study reveals that elevation of RBM20 disturbed lysosome acidification in doxorubicin induced cardiotoxicity via the alternative splicing of Atp6v0a1, leading to heart failure.JUN-Rbm20 upregulattion promoted DICRBM20 alternative spliced atp6v0a1