Role Of MYBPC3 Gene Mutation In HCM Myocardial Fibrosis

Hypertrophic cardiomyopathy(HCM)is an autosomal dominant genetic disease caused by single gene mutation.Its pathological features mainly include myocardial cell hypertrophy,disorder of arrangement,myocardial fibrosis and connective tissue hyperplasia.The incidence of HCM is 0.2%,which is the main cause of Sudden Cardiac Death(SCD)in adolescents and athletes.It can occur at any age group and has significant familial aggregation.Familial onset accounts for 50% of all patients,and the annual mortality rate is about 1%~2%.A large number of evidence revealed MYBPC3 is the most frequently mutated HCM gene,representing about 40~50% of all HCM mutations.The mutation forms of MYBPC3 gene are varied,mainly including nonsense mutations,deletion or insertion mutations,and splice site mutations.The MYBPC3 gene encodes cardiac myosin binding protein C(c My BP-C),which is located in the transverse bridge area of the two ends of the thick filaments.It is an important part of striated muscle.Its main function is to regulate adrenal hormone and connect the A band of myosin molecules and sarcomeres to complete the response of the heart and stabilize the structure of sarcomeres.Myocardial fibrosis(MF)has been proved to be an important factor and determinant in the pathogenesis of HCM.MF is common pathological manifestation of alterations in cardiac structure and function,mainly manifested by the proliferation of cardiac fibroblasts(CFs)and excessive deposition of collagen in myocardial tissue.Fibrogenesis is considered to be the repair response of the body after injury to maintain the structural integrity of tissues and organs.However,excessive or uncontrollable repair process can lead to organ fibrosis.In severe cases,the development of fibrosis destroys the normal structure and function,posing a serious threat to human health.However,there is no evidence to show the role of MYBPC3 gene mutation in HCM myocardial fibrosis.This study aims to investigate the relationship between MYBPC3 gene mutation and myocardial fibrosis in HCM and its related molecular mechanisms,including three parts as follow:(1)Model pigs imitating clinical MYBPC3 point mutation were established.Single-base gene editing system and somatic nuclear transfer(SCNT)were used to imitate the missense mutation R495 Q of MYBPC3 gene found in 1998 to prepare genemodified HCM pigs.The genotype and phenotype results of point mutation pigs showed that R495 Q gene mutation significantly reduced the expression level of MYBPC3 in CFs;significantly increased the levels of myocardial injury-related indicators(hs-TNT and PCIP),fibrosis markers TGF-?1 and inflammatory factors,and severe inflammatory cell infiltration occurred in myocardial tissue.R495 Q gene mutation significantly increased the m RNA and protein expression levels of TGF-?1,COL1A1 and ?-SMA in myocardial tissue.Masson staining showed that mutations led to severe myocardial fibrosis phenotype.Moreover,R495 Q gene mutation significantly increased lactic acid levels in serum and myocardial tissue.(2)To explore the effect of MYBPC3 gene mutation on fibroblast activation.Firstly,it was determined that MYBPC3 gene was expressed in porcine cardiac fibroblasts(PCFs),mouse cardiac fibroblasts(MCFs),mouse embryonic fibroblasts(NIH-3T3)and human cardiac fibroblasts(HCFs).CRISPR/Cas9 gene editing system was used to establish MYBPC3 gene knockout fibroblasts to detect the effects of gene deletion on cell proliferation,cycle,apoptosis and migration.The results showed that the deletion of MYBPC3 gene significantly improved the proliferation ability of fibroblasts,accelerated the cell cycle process,reduced the number of apoptotic cells,and enhanced the cell migration ability.The expression of fibrosis markers after fibroblasts activation was detected,and the results showed that the deletion of MYBPC3 gene significantly increased the expression levels of COL1A1,?-SMA and TGF-?1 in fibroblasts,and the content of TGF-?1 in fibroblasts was significantly increased.(3)Molecular mechanism of myocardial fibrosis induced by MYBPC3 gene deletion.The levels of lactic acid in serum and tissue of MYBPC3-R495 Q point mutation pigs were significantly increased.In order to further determine the glycolysismediated MYBPC3 gene deletion leading to myocardial fibrosis,the levels of glucose,lactic acid and ATP in fibroblasts were detected.The results showed that MYBPC3 gene deletion significantly accelerated glucose consumption,increased lactic acid content,and promoted intracellular ATP synthesis.At the same time,MYBPC3 gene deletion significantly up-regulated the m RNA and protein expression levels of key enzymes and important genes in glycolysis pathway;significantly up-regulated the expression of HIF-1? enhancing GLUT1,PFK,LDHA transcription,which is consistent with the enhanced glycolysis flux.Inhibition of glycolysis significantly reduced the expression of fibrosis marker genes COL1A1 and ?-SMA up-regulated by MYBPC3 deletion,but did not affect the expression of TGF-?1.Studies have shown that the interaction between HIF-1? and TGF-?1 promotes fibrosis.The results of this study showed that in MYBPC3-deficient fibroblasts,HIF-1? inhibitor Oltipraz significantly reduced the expression of HIF-1?,glycolysis pathway and fibrosis marker genes,but did not affect the expression of TGF-?1.TGF-?1 inhibitor SB-431542 significantly inhibited the expression of HIF-1?,glycolysis pathway and fibrosis marker genes.It is known that TGF-?1 and NF-?B are involved in the occurrence of liver fibrosis.The results of this study showed that the phosphorylation of p65 increased in MYBPC3-deficient fibroblasts,and the NF-?B signaling pathway was activated.PDTC inhibited the NF-?B signaling pathway and significantly reduced the expression of TGF-?1 and HIF-1?,while SB-431542 did not inhibit the NF-?B signaling pathway.In summary,we used gene editing technology to imitate the clinical MYBPC3R495 Q gene mutation in model pigs,which proved that this gene mutation led to myocardial fibrosis.In vitro fibroblasts,it was clarified that MYBPC3 gene mutation activated NF-?B/ TGF-?1/ HIF-1?/ aerobic glycolysis signal cascade,promoted the activation of fibroblasts,and led to fibrosis phenotype.