Rbm24 Regulates Cardiac Sarcomere Assembly Contributing To Dilated Cardiomyopathy

ObjectiveTo investigate the regulation mechanism of Rbm24 in maintaining cardiac structure and function after birth,and to explore the relationship between Rbm24 deficiency and dilated cardiomyopathy.In addition,analysis of the phosphorylation mechanism of Rbm24 protein and the relationship between Rbm24 protein stability and cardiac sarcomere assembly.It provides a more comprehensive cognition for RNA binding protein mediated RN A splicing and the pathogenesis of dilated cardiomyopathy,uncovers a potential novel pathway to cardiomyopathy through modulating the Stk38/Rbm24 protein activity.MethodsFirst,the Cre-loxP system was used to construct Flper mice and mated with cardiac-specific MHC-cre mice to obtain Rbm24 heart-specific knockout mice.The knockout efficiency of knockout and heterozygous mouse Rbm24 was analyzed at the RNA level and protein level,respectively.In addition,the structural changes of the heart of KO mice were analyzed by HE staining,Masson staining,etc.and the heart disease type was comprehensively judged by combining qPCR and cardiac UTE.RNA-seq was used to further analyze the changes in alternative splicing caused by Rbm24 deletion and to perform GO analysis.Those reported cardiomyopathy-related genes and sarcomere-associated genes were selected for RT-PCR specific validation.Respectively,both HL-1 cell and primary cardiomyocytes were knockdown Stk38.RT-PCR analysis of alternative splicing sarcomere genes,Western blot and immunofluorescence staining were used to detect sarcomere assembly.In addition,the recovery of sarcomere assembly was analyzed by overexpression of Rbm24 in shStk38 cells.In vitro kinase assays were performed to analyze the phosphorylation level of Rbm24.Meanwhile,phosphorylation and stability of Rbm24 protein were analyzed by using OA/BAPTA-AM treatment coupled with immunoprecipitation technique,respectively.ResultsHere,we report that Rbm24 was ablated in heart of knockout mouse at 5 days old,and all died before 2 months of age.Then,conditional knockout of Rbm24 leads to rapidly progressive dilated cardiomyopathy,heart failure,and postnatal lethality.In-depth analysis of the RNA-seq shows that the major alternative splicing mode regulated by Rbm24 is the exon inclusion(exclusion),and the role of Rbm24 is to suppress the loss of exons and promote its retention.Rbm24 is an inhibitor of alternative splicing.The biological function classification analysis of the regulated genes revealed that these genes are mainly involved in the formation of myocytes,the assembly of sarcomere,the rhythmic contraction of myocardium.Those results suggest that Rbm24 regulates alternative splicing of genes included Titin,involved in multiple aspects of cardiac function and participates in postnatal remodeling of the heart by controlling key developmentally regulated splicing switches.Knockout of Rbm24 abnormally modulates changes in sarcomere resulting from altered splicing of genes associated with heart disease,eventually leading to the development of dilated cardiomyopathy.Stk38 knockdown resulted in decreased Rbm24 protein level in cardiomyocytes.Further studies shown that Rbm24 is a phosphoprotein.Using Stk3 8 kinase inhibitor or activator showed that Rbm24 protein stability was regulated in a kinase activity-dependent manner.Deficiency of Stk3 8 caused reduction of sarcomere proteins and disarrangement of sarcomere,suggesting that Stk3 8 is essential for Rbm24 to regulate sarcomere assembly.ConclusionRNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy.Meanwhile,Stk38 modulates Rbm24 protein stability to regulate sarcomere assembly in Cardiomyocytes.This furthers our understanding of the regulatory mechanism of cardiac sarcomere assembly in both physiologic and pathologic contexts and uncovers a potential novel pathway to cardiomyopathy through modulating the Stk38/Rbm24 protein activity.