Mechanism Of LAMA2 Regulation In Arrhythmogenic Right Ventricular Cardiomyopathy Using An Induced Pluripotent Stem Cell Derived Cardiomyocyte Model

Objective:To study the cellular function/signaling pathways based on the construction of an induced pluripotent stem cells(i PSCs)-derived cardiomyocyte model,and to further explore the potential pathogenic mechanism of a newly discovered gene mutation of LAMA2 in the pathogenesis of arrhythmogenic right ventricular cardiomyopathy(ARVC).Materials and methods:The blood samples of ARVC patients and their parents were evaluated by whole exome sequencing and gene harmfulness analysis to screen possible pathogenic gene mutation sites.Patient-specific i PSCs were then generated from peripheral blood mononuclear cells of patients.After that,the stemness and multiple differentiation potential of i PSCs were identified by using immunohistochemistry,quantitative reverse-transcriptase polymerase chain reaction(q RT-PCR),flow cytometry,alkaline phosphatase staining and in vivo three germ layer development test in nude mice.An oriented inducement of i PSCs into cardiomyocytes was performed by Gi Wi protocol.Furthermore,Gene Ontology(GO)-based functional enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway analysis to study possible pathogenic signaling pathways were performed in this cardiomyocyte line and normal cardiomyocyte line by RNA-seq technique.After that,the screened signaling pathways were verified by q RT-PCR.In addition,Western blot was performed to measure and analyze the expression levels of LAMA2 and related proteins,fibrosis and adipose signaling pathways-related terminal proteins.Results:According to the bioinformatics analysis of whole exome sequencing results in ARVC patients,a total of 7 candidate gene mutation sites were screened,among which only LAMA2(NM＿001079823: exon61:c.G8830A)mutation site was found to be associated with ARVC.Further direct sequencing was performed to verify the accuracy of LAMA2 mutation site.Sendai virus carrying transcription factors(OCT3,SOX2,KLF4 and c-MYC)was used to reprogram peripheral blood mononuclear cells of patients and differentiate these cells into i PSCs.The results of karyotype analysis of the i PSCs differentiated in our experiment were consistent with those of the patients enrolled.q RT-PCR and immunofluorescence were then performed to detect cell stemness biomarkers(AURKB,Ki67,SSEA4,OCT4,SOX2,NANOG,KLF4,etc.)at m RNA and protein levels.Corresponding results showed that i PSCs had good stemness.Meanwhile,the i PSCs had high purity based on the detection of stem cell-specific markers(TRTA-1-60,SSEA4 and OCT4)by flow cytometry.The positive alkaline phosphatase staining reaction indicated that the i PSCs were in the initial state of undifferentiated embryonic-like stem cells.The results of in vivo teratoma differentiation experiment showed that the i PSCs could differentiate into several histocytes.Furthermore,i PSCs were induced into cardiomyocytes by Gi Wi protocol,and immunofluorescence staining confirmed that the induced cells expressed c TNT and myl2,two specific markers of cardiomyocytes.At the same time,normal human peripheral blood mononuclear cells were reprogrammed successfully to differentiate into i PSCs reversely according to the above protocol,after which oriented inducement of the i PSCs into cardiomyocytes was performed based on Gi Wi protocol,which was set as the control group.The cardiomyocytes.of ARVC group and control group were compared by RNA-Seq sequencing.Afterwards,according to the pathological characteristics of ARVC(cell apoptosis,inflammation,intracellular calcium signals,fibrosis,fatty degeneration and cell junction),functional gene sets were established based on the sequencing results,followed by GO and KEGG gene enrichment analysis.The results of GO and KEGG gene enrichment analysis indicated that compared with control group,the expression levels of genes involved in the above signaling pathways was significantly increased in ARVC group.It was speculated that the above signaling pathways were activated in this group of cardiomyocytes.For further confirmation of the results of gene enrichment analysis,q RT-PCR was conducted to analyze the m RNA expression of genes in each node of the above signaling pathways.As revealed by the results:(1)Apoptosis signaling pathway: the expression levels of CACP3,CACP8,BAX,BCL2 and CAPN2 were significantly upregulated in ARVC group;(2)inflammatory response signaling pathway: there were obvious increase in the expression levels of ICAM-1,TNFα,IL1 and IL6;(3)Calcium signaling pathway: a decreased trend was found in the expression of RYR2 in ARVC group;(4)Fibrosis signaling pathway related to extracellular matrix: the expression levels of VCL,ACTA2,CTCF,ITGB2,ITGB1 and TLN1 were remarkably increased in ARVC group,while those of PTK2 and ITGA2 were decreased when compared with those in control group;(5)Fatty degeneration related signaling pathway: there were obvious upregulation of PPARG,CEBPB,GATA3,ADIPOQ,LEP and FABP4 expression levels,while evident downregulation of GATA2,GATA4 and WNT1 expression levels in ARVC group;(6)Cell junction related pathway: the expression levels of JUP,ACTN1,ACTN4,ACTB,ACTG1,DSC2,DSG2 and GJA1 were detected to be highly increased while those of CTNNA2,CTNNA3 and CDH2 were decreased in ARVC group when compared with control group.The results of q RT-PCR described above were consistent with those of GO and KEGG gene enrichment analysis.Moreover,compared with the cardiomyocytes in control group,the protein expression of LAMA2 was reduced in the cardiomyocytes of ARVC group,but accompanied by a decrease in the expression levels of its related proteins Intergin-β1 andβ-Dystroglyean.Besides,the expression of terminal protein CTCF in fibrosis signaling pathway was increased,and that of terminal protein Adiponectin protein in fatty degeneration related signaling pathway was increased in the cardiomyocytes of ARVC group.In addition,based on the results of immunofluorescence,in ARVC group,cardiomyocytes were positively stained by using both SMA and Vimentin(myofibroblast markers),as well as c TNT(cardiomyocyte marker)antibodies.It suggested that the cardiomyocytes were differentiated into fibroblasts.Conclusion:We speculate that LAMA2 mutation may be a new pathogenic gene of ARVC.The pathogenic mechanism of ARVC caused by LAMA2 mutation:(1)The activation of the apoptosis signal pathway of cardiomyocytes in ARVC group may be related to the imbalance of ECM protein ratio in cardiomyocytes of ARVC group caused by LAMA2 mutation(LAMA2 protein decreased significantly,and DAG1 and ITGB1 gene and protein expression increased significantly).(2)LAMA2mutations increased the expression of PG,thereby inhibiting the Wnt/-catenin signaling pathway,which leads to ARVC.(3)LAMA2 mutations can lead to myocardial fibrosis by inhibiting the Wnt/-catenin signaling pathway and activating the TGF- pathway,and we observed the differentiation of cardiomyocytes carrying LAMA2 mutations into fibroblasts.(4)LAMA2 mutations can lead to myocardial fatification by inhibiting the Wnt/-catenin signaling pathway and It also indicates that cardiomyocytes carrying LAMA2 mutations may differentiate into adipocytes.(5)The m RNA expression of Ry R2 receptor on mitochondria in the cardiomyocytes of ARVC group was significantly decreased,which may lead to arrhythmia in this ARVC patient with LAMA2 mutation.