Sorafenib induces cardiotoxicity through RBM20 mediated alternative splicing of sarcomeric and mitochondrial genes

Tyrosine kinase inhibitors (TKIs), as a class of small-molecule drugs that exert anti-tumor effects by inhibiting tyrosine kinase-catalyzed phosphorylation, have been used in the treatment of various cancers. Sorafenib, as a multi-targeted TKI drug, is the first-line treatment for advanced renal cell carcinoma and unresectable hepatocellular carcinoma. However, sorafenib has repeatedly been reported to cause cardiac events in patients without a history of heart diseases during clinical use, indicating that it has cardiotoxicity. Alternative splicing of cardiac contraction-related genes happens during heart development and cardiac diseases, and is critical for heart function. However, whether alternative splicing plays a role in drug-induced cardiotoxicity remains unexplored. RBM20 is an important cardiac-specific splicing factor, mutations of which cause dilated cardiomyopathy or other cardiac dysfunctions. Rbm20 also mediates alternative splicing of genes essential for heart contraction, which is often negatively affected in drug-induced cardiotoxicity. Existing studies do not fully explain the mechanism of sorafenib cardiotoxicity, and none of the relationship between cardiotoxicity of sorafenib and alternative splicing mediated by tissue-specific splicing factors, such as Rbm20, have been reported. In order to explore whether cardiac-specific alternative splicing plays a role in sorafenib-induced cardiotoxicity, we establish both cell and animal models of cardiotoxicity, and obtain the following results: (1) By constructing a rat animal model administered with sorafenib, we find that sorafenib causes abnormal cardiac function in rats, and the genes that undergo alternative splicing in rat hearts are related to cytoskeleton of actin; (2) Alternatively spliced genes induced by sorafenib in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are enriched in sarcomere, actin filament, calcium transient regulation, mitochondria, all of which are critical for cardiac contraction. These genes are associated with dilated cardiomyopathy, hypertrophic cardiomyopathy and other cardiomyopathy; (3) Sorafenib induces a decrease in the expression of cardiac-specific splicing factor RBM20; (3) Many genes whose splicing are altered by sorafenib overlap with Rbm20 targets, indicating that sorafenib may affect alternative splicing through Rbm20; (4) Sorafenib induces pathogenic alternative splicing of FHOD3, which is a RBM20 target gene and participates in myocardial sarcomere formation. Sorafenib also affects alternative splicing of SLC25A3, which encodes a phosphate transporter on the mitochondrial inner membrane and regulates ATP synthesis; (5) Enhancing the expression of RBM20 rescues the cardiotoxicity of sorafenib by reducing apoptosis and increasing ATP levels, which is mediated by reversing the alternative splicing of FHOD3 and SLC25A3 induced by sorafenib. This paper uncovers that sorafenib reduces the expression of RBM20 to cause pathogenic alternative splicing of genes related to myocardial sarcomere and energy mechanism, resulting in abnormal myocardial function. Increasing the expression of RBM20 reverses the alternative splicing of FHOD3 and SLC25A3 associated with cardiac sarcomeres and mitochondria respectively, rescuing the cardiotoxicity of sorafenib.酪氨酸激酶抑制剂（TKIs）是一类通过抑制酪氨酸激酶催化的磷酸化反应发挥抗肿瘤作用的小分子药物，已被用于多种癌症的治疗。索拉非尼作为一款多靶点TKIs药物，是晚期肾细胞癌与不可切除肝细胞癌的一线治疗用药。然而，临床使用中多次有报道称，无心脏病史的患者在使用索拉非尼后会出现心脏不良事件，提示其具有心脏毒性。 心脏收缩相关基因的可变剪接（alternative splicing）发生于心脏发育与心脏疾病进程中，对心脏功能至关重要。但可变剪接是否参与药物诱导的心脏毒性，目前仍未被阐明。RBM20是一种重要的心脏特异性剪接因子，其突变可引发扩张型心肌病或其他心脏功能异常；RBM20同时可介导心脏收缩必需基因的可变剪接，而这类剪接过程在药物诱导的心脏毒性中常受到破坏。现有研究尚未完全阐明索拉非尼心脏毒性的作用机制，且目前尚无研究报道索拉非尼的心脏毒性与RBM20等组织特异性剪接因子介导的可变剪接之间的关联。 为探究心脏特异性可变剪接是否参与索拉非尼诱导的心脏毒性，本研究构建了心脏毒性细胞模型与动物模型，并获得如下结果： (1) 通过构建索拉非尼给药的大鼠动物模型，本研究发现索拉非尼可导致大鼠心脏功能异常，且大鼠心脏中发生可变剪接的基因与肌动蛋白细胞骨架相关； (2) 索拉非尼在人类诱导多能干细胞衍生心肌细胞（hiPSC-CMs）中诱导的可变剪接基因显著富集于肌节、肌动蛋白丝、钙瞬态调控、线粒体等与心脏收缩功能密切相关的范畴，这些基因与扩张型心肌病、肥厚型心肌病等多种心肌病相关； (3) 索拉非尼可下调心脏特异性剪接因子RBM20的表达； (3) 索拉非尼诱导可变剪接改变的基因与RBM20的靶基因存在大量重叠，提示索拉非尼可能通过RBM20影响可变剪接过程； (4) 索拉非尼可诱导RBM20靶基因FHOD3发生致病性可变剪接，而FHOD3参与心肌肌节的形成；同时索拉非尼还可影响SLC25A3的可变剪接，该基因编码线粒体内膜上的磷酸转运体并参与ATP合成调控； (5) 过表达RBM20可通过逆转索拉非尼诱导的FHOD3与SLC25A3可变剪接，减少细胞凋亡并提升ATP水平，从而缓解索拉非尼的心脏毒性。 本研究揭示，索拉非尼通过下调RBM20的表达，引发心肌肌节相关基因与能量代谢相关基因的致病性可变剪接，最终导致心肌功能异常；而过表达RBM20可分别逆转与心肌肌节、线粒体相关的FHOD3与SLC25A3的可变剪接，从而改善索拉非尼诱导的心脏毒性。