Establishment And Mechanism Of Mouse Model Of Myocardial Hypertrophy With MYBPC3 Gene Frameshift Mutation

Objective:This study is based on myocardial hypertrophy due to the MYBPC3 gene frameshift mutation previously identified in patients with familial hypertrophic cardiomyopathy,but the underlying disease mechanism is unknown.To this end,we established a mouse model of myocardial hypertrophy carrying MYBPC3 frameshift mutation site,in order to explore the occurrence and development mechanism of myocardial hypertrophy and provide a new target for the precise treatment of HCM.This study consists of three parts,the first part is to establish a mouse model of myocardial hypertrophy carrying MYBPC3 gene frameshift mutation and evaluate whether the model is successfully established from multiple dimensions.The second part analyzes the transcription profile of mouse myocardial tissue by transcriptome sequencing to screen for differential genes and signaling pathways that may be related to the process of myocardial hypertrophy.The third part uses Western blot technology to verify the key proteins of AKT-mTOR and CaMKK2-AMPK-FOXO3A signaling pathway,so as to preliminarily explore the role and mechanism of MYBPC3 gene frameshift mutation in myocardial hypertrophy.Methods:1.Construction of transgenic mice.A transgenic mouse model with MYBPC3^{（c.3636del T）}frameshift mutation locus was constructed using CRISPR/Cas9 gene editing technology.The genotypes of the mice were identified using agarose gel electrophoresis and Sanger sequencing.2.Evaluation of myocardial hypertrophy models.Western blot technique and immunofluorescence were used to detect the expression of c My BP-C in myocardial tissues.A comprehensive pathological evaluation of the mouse heart was done,including gross cardiac assessment as well as staining of myocardial tissue sections.Hematoxylin-eosin（HE）staining was used to visualize the nucleus and cytoplasm;Masson staining was used for the detection of collagen fibers;and Wheat Germ Agglutinin（WGA）staining was used to assess the cross-sectional area of cardiac myocytes.The m RNA expression levels of hypertrophy biomarkers（BNP,ANP,β-MHC）and fibrosis markers（CollagenⅠα,CollagenⅢα,CTGT）in myocardial tissue were detected using Real-time q PCR.The expression of SERCA2 and RYR2 proteins related to calcium transport on the sarcoplasmic reticulum,were also detected by immunohistochemistry.Fresh myocardial tissue from mice was quickly fixed in an electron microscope fixative to observe the ultrastructure of sarcomere,mitochondria,and the presence or absence of autophagosomes in myocardial tissue under scanning electron microscopy.3.Transcriptome sequencing analysis of mouse myocardial tissues.Total RNA from mouse myocardial tissue was extracted and reverse transcribed into c DNA for transcriptome sequencing（RNA-Seq）.During differentially expressed gene detection,gene expression was normalized to FPKM,and the screening criteria were Fold Change of expression difference≥2 and false discovery rate FDR<0.05 for sequencing data.4.Preliminary exploration of the mechanism of MYBPC3 gene frameshift mutation causing myocardial hypertrophy.The expression levels and activity changes of AKT-mTOR/CaMKK2-AMPK-FOXO3A signaling pathways in myocardial tissues were examined by Western blot technique.Results:1.The mice were identified by agarose gel electrophoresis and Sanger sequencing as wild-type(MYBPC3^+/+,WT),heterozygous(MYBPC3^+/-,Het),homozygous(MYBPC3^-/-,Hom).Western blot,Real-time q PCR,and immunofluorescence results showed that the m RNA expression level of MYBPC3 was significantly reduced in homozygous(MYBPC3^-/-,Hom)mice,the m RNA expression level of MYBPC3 was significantly reduced and c My BP-C was not expressed.c My BP-C expression level in heterozygous(MYBPC3^+/-,Het)mice was about 50%of that in wild-type(MYBPC3^+/+,WT)mice.2.Mutant(MYBPC3^+/-,Het;MYBPC3^-/-,Hom)mice showed dilatation of the cardiac chambers and significant thickening of the ventricular wall,with the most pronounced in homozygous(MYBPC3^-/-,Hom)mice.Pathological staining showed disorganized myonodular arrangement,increased myocardial cell volume,inflammatory cell infiltration and more blue collagen fibers in the myocardial tissue in homozygous(MYBPC3^-/-,Hom)mice,followed by heterozygous(MYBPC3^-/-,Hom)mice.The m RNA expression levels of the major markers of myocardial hypertrophy（BNP,ANP,β-MHC）and myocardial fibrosis markers（CollagenⅠα,CollagenⅢα,CTGT）were significantly higher in homozygous(MYBPC3^-/-,Hom)and heterozygous(MYBPC3^+/-,Het)mice compared with wild-type(MYBPC3^+/+,WT)mice.Immunohistochemical results showed that the expression of SERCA2 was significantly lower in homozygous(MYBPC3^-/-,Hom)and heterozygous(MYBPC3^+/-,Het)mice than in wild-type(MYBPC3^+/+,WT)mice,while the expression level of RYR2 was not significantly different.Under scanning electron microscopy,homozygous(MYBPC3^-/-,Hom)and heterozygous(MYBPC3^+/-,Het)mice showed disordered arrangement of sarcomere,significantly increased number of mitochondria,swelling and degeneration,ridge rupture,and even the presence of autophagy lysosomes.3.The transcriptome sequencing results showed that a total of 724 DEGs were differentially expressed in homozygous mice(MYBPC3^-/-,Hom)compared with wild-type mice(MYBPC3^+/+,WT),and 22 DEGs were differentially expressed in heterozygous mice(MYBPC3^+/-,Het).The results of KEGG pathway enrichment bubble map and GO functional analysis suggested that the differentially expressed genes in homozygous(MYBPC3^-/-,Hom)mice mainly regulated the signaling pathways of PI3K-AKT,myocardial hypertrophy,fibrosis,autophagy,Ca²⁺signaling pathway,and matrix remodeling.4.AKT-mTOR signaling pathway was significantly upregulated in homozygous(MYBPC3^-/-,Hom)mice;meanwhile,CaMKK2-AMPK-FOXO3A signaling pathway was also activated.Conclusions:For the first time,transgenic mice carrying MYBPC3^{（c.3636del T）}frameshift mutation sites were established,which showed significant myocardial hypertrophy,and the mouse model of myocardial hypertrophy was successfully evaluated by systematic evaluation.In mice with myocardial hypertrophy,homozygous mice did not express the c My BP-C protein,and the expression of the heterozygous type was about half that of the wild type,supporting the idea of haplodose inadequacy.In addition,there were differential gene expressions such as AKT-mTOR,CaMKK2-AMPK-FOXO3A,myocardial hypertrophy,fibrosis,calcium ion homeostasis,and autophagy in mouse models of myocardial hypertrophy,which were verified by Western bolt,indicating that the two signaling pathways of AKT-mTOR and CaMKK2-AMPK-FOXO3A were involved in the regulation of myocardial hypertrophy.