Isoliquiritin targeting m5C RNA methylation improves mitophagy in doxorubicin-induced myocardial cardiotoxicity

Abstract

Background: Doxorubicin (DOX)-induced myocardial cardiotoxicity (DIC) severely limits its clinical application, and there is no optimal therapeutic agent available. Recent studies revealed that activation of BNIP3-mediated mitophagy and the inhibition of m5C RNA methylation played a crucial role in DIC. Isoliquiritin (ISL) has remarkable cardiac protective effect. But its potential mechanisms against DIC still remains unknown.

Purpose: To investigate the therapeutic effect and potential mechanism of Isoliquiritin(ISL) on doxorubicin(DOX)-induced myocardial cardiotoxicity(DIC).

Methods: Bioinformatics analyses and network pharmacology were carried out to identify effective target of ISL against DIC. Molecular docking and surface-plasmon resonance (SPR) were used to confirm the predict. The mechanism of ISL regulating mitophagy through M5C methylation to improve DIC was demonstrated in vitro and in vivo experiments. The methylation modification was verified by MeRIP-qPCR. Cell model of DIC was constructed to evaluate mitochondrial function by measuring cell viability, myocardial enzyme level, mitochondrial quality, mCherry-EGFP analysis and TEM morphometry with the application of mitophagy inhibitor (Baf A1) and inducer (CCCP). Myocardial injury in mice with DIC was assessed by survival rate, myocardial enzyme level, HE staining, echocardiography and detection of mitophagy markers.

Results: The decreased level of m5C writer TRDMT1 and mitochondrial marker (BNIP3) were chosen for the research. After the docking and SPR verification between ISL and TRDMT1, the improvement of ISL on TRDMT1 and TRDMT1-associated m5C level of BNIP3 was identified. In vitro and in vivo experiments showed that the cardiac markers, heart function, and mitochondrial function were recovered after ISL application. Meanwhile, the results manifested that there was blocked autophagy flow indicated by mCherry-EGFP analysis, then the suppressed mitophagy caused the mitochondria damage associated cell death. ISL could alleviate the autophagy block, and Baf A1 couldn't influnce the ISL effect. Compared to CCCP group, Mitochondrial maker TOMM20 significantly elevated treated with both CCCP and DOX, indicating that DOX impaired mitophagy for clearing damaged mitochondrial proteins. After ISL treated, a higher level of co-localization between mitochondrial and BNIP3 was observed, inducing restoration of mitochondrial function.

Conclusion: This study showed that ISL may exert cardioprotective role restoring BNIP3-mediated mitophagy by targeting TRDMT1 to alleviate DOX-induced macro-autophagy-dependent protein homeostasis and acquired blocking of mitophagy, providing a new idea for the clinical treatment of DIC.

Keywords: BNIP3; Cardiotoxicity; Doxorubicin; Isoliquiritin; Mitophagy; m5C RNA methylation.