The Role And Mechanism Of M~6A Recognition Protein YTHDC1 In Regulating Dilated Cardiomyopathy

BACKGROUNDDilated Cardiomyopathy(DCM)is one of the most common causes of heart failure(HF)and it is the leading indication for heart transplantation.It is characterized by left or bicentricular ventricular chamber enlargement with impaired systolic dysfunction or not.There is ample data on the familial aggregation of DCM(30-50%),suggesting a relevant genetic involvement.Over 60 genes have been reported involving in the regulation of dilated cardiomyopathy and participating in encoding the sarcomere,cytoskeleton,nuclear envelope,transcriptional pathways,and mitochondrial proteins.Therefore,regulating the progression of dilated cardiomyopathy by gene to ameliorate or treat heart failure caused by dilated cardiomyopathy provides a novel therapeutic strategy.However,the genetic molecules and underlying mechanisms driving the progression of dilated cardiomyopathy have only been partially elucidated.N~6-methyladenosine(m~6A)is the most abundant post-transcriptional modification in eukaryotic mRNA and is crucial for various biological processes,regulating the pathological process of multiple diseases.This modification is dynamically regulated by"writing"by m~6A methyltransferase METTL3-METTL14-WTAP compounds and removed by m~6A demethylase molecules such as FTO and ALKBH5.As m~6A‘reader’proteins,YTH domain-contained proteins are the major protein family participating in recognizing and binding the m~6A modification.Recent studies have revealed that m~6A methyltransferase METTL3 and demethylase FTO play important roles in cardiac remodeling.However,the function of the m~6A reader protein in the heart remains unknown.Our study aims to explore the bio-functional role of m~6A‘reader’proteins in heart.METHODS AND RESULTSTo determine the role of m~6A‘reader’proteins in the heart,we generated four genetic knockout mice(including Ythdf1-KO,Ythdf2-c KO,Ythdf3-KO and Ythdc1-c KO)to allow assessment the role of each m~6A‘reader’protein family member in cardiac function.Consistent with pathological observations,we found the left ventricular ejection fraction decreased from 84.8±3.37%to 28.1±4.41%and the end-diastolic LV internal diameter increased from 2.73±0.17 mm to 4.69±0.24 mm,suggesting that cardiac-specific Ythdc1 knockout mice developed typical DCM characterized by left ventricular chamber enlargement and systolic dysfunction.Meanwhile,we also found that the contraction of single cardiomyocytes dramatically weakened.By using multiple RNA sequences,we identified 42 changed downstream target genes in Ythdc1 knockout mice.Finally,we confirmed that Ythdc1-deficiency mice developed DCM as a result of altered Titin RNA splicing.Ythdc1-deficiency in cardiomyocytes statistically increases the ratio of Titin N2BA to N2B.The disruption of intracellular myofilament structures caused by this change in Titin mRNA splicing in Ythdc1-deficient cardiomyocytes resulted in DCM.CONCLUSIONOur study demonstrates that the m6A reader protein Ythdc1 participates in the development of DCM by regulating the alternative splicing of Titin mRNA,providing a novel potential target for the treatment of DCM.