Induced Pluripotent Stem Cell-derived Cardiomyocytes As A Model For Danon Disease And Insights Into The Pathogenesis Of Danon Disease Based On A Multi-omics Approach

Background:Danon disease(DD)manifested by cardiomyopathies has high incidence and mortality but no effective treatments.Induced pluripotent stem cell-derived cardiomyocytes(iPSC-CMs)have been extensively applied to studies on inherited cardiomyopathies,including DD,because induced pluripotent stem cells(iPSCs)can produce an unlimited number of cardiomyocytes containing donor genetic information.Despite the current knowledge of DD regarding the phenotypes and dysfunction of cardiomyocytes.further studies are needed to investigate the mechanisms responsible for the development and progression of the disease and identify effective interventions.Objective:Aiming to investigate potential mechanisms responsible for the phenotypes of DD,this study modeled DD by differentiating patient-specific iPSC lines into CMs for multi-omic sequencing and performed a systematic analysis of the results produced by the multi-omics approach.Methods:Peripheral blood mononuclear cells(PBMCs)from DD patient were reprogrammed into iPSCs and subsequently differentiated into iPSC-CMs.The iPSCs were examined by morphology,karyotype analysis,teratoma formation assay,flow cytometry,confocal fluorescence microscopy,and real-time qPCR.The iPSC-CMs were identified based on morphology,spontaneous and independent beating records,confocal fluorescence microscopy,autophagosome and autophagic flux monitoring,and glycogen storage assessment.Transcriptomic data were subject to analysis of differentially expressed genes,enrichment analysis,protein-protein interaction(PPI)network analysis and comparative analysis with the datasets available in open-source databases.Proteomic data were assessed by analysis of differential proteins,enrichment analysis,PPI network analysis and association analysis with the transcriptomic data.Phosphoproteomic data were evaluated by analysis of differential phosphorylation site,enrichment analysis,protein motif analysis,kinase activity prediction,and kinase regulatory network analysis.Results of the multi-omics analyses were verified by western blotting,cellular metabolism assay,and patch-clamp electrophysiology.Results:IPSCs of genetic stability and morphological uniformity and carrying DD-associated pathogenic mutations were modeled to differentiate into spontaneously beating iPSC-CMs in which CM-specific structural proteins were expressed,LAMP2 protein expression was significantly reduced,phenotypic characteristics such as autophagosome accumulation and autophagic flux blockage were detected,and glycogen accumulation was unremarkable.In the transcriptomic data analysis,248 differentially expressed genes(DEGs)were identified,and the genes with upregulated expression in the DD group were found to be associated with transmembrane transport of simple sugars and ions,lipid metabolism,and extracellular matrix(ECM)assembly,while those with downregulated expression were shown to have associations with DNA transcription and histone modifications.A total of 114 DEGs were obtained from the datasets downloaded from open sources and merged with those identified in the transcriptomic data analysis.Through a careful analysis,it was noted that there were only two co-upregulated genes,including RAB31 and FLRT2.In the proteomic data analysis,166 differentially expressed proteins(DEPs)were identified,including 20 upregulated proteins and 20 downregulated proteins in the DD group,with the upregulated proteins being associated with glycolysis and the downregulated ones related to lysosomes;the PPI network analysis indicated that there were 20 upregulated and 20 downregulated proteins;the transcriptome-wide association analysis suggested the presence of only two co-upregulated proteins,namely HSPA2 and CMBL.In the phosphoproteomic data analysis,261 phosphorylation sites on 209 proteins were significantly upregulated,while 81 phosphorylation sites on 74 proteins were substantially downregulated in the DD group.Proteins with upregulated modification sites were shown to have strong correlations with the regulatory effects on the action potentials of ventricular myocytes,and those with downregulated modification sites were closely associated with the regulation of guanosine triphosphatase activity.According to the kinase activity prediction and kinase regulatory network analysis,11 kinases appeared to possess critical regulatory properties.Based on the verification results,DD iPSC-CMs exhibited a significant increase in the ALDOA,PGAM2,and HSPA2 expression levels;changes in the VTI1B and SGSH expression levels were unremarkable;the levels of glycolysis and cellular oxygen uptake were elevated significantly;the absolute values of the action potential amplitude(APA)and the mean diastolic potential(MDP)rose to higher levels;the calcium current density appeared to decrease continuously.Conclusions:Modeling DD with specific CM phenotypes through identification of iPSC-CM phenotypes in DD patients provides a reliable platform for studying the mechanisms of or therapy for DD.Considering the substantial heterogeneity in transcriptomic data and between transcriptomic and proteomic data,extensive data are required to support the interpretation of the analytical results.According to the proteomic data analysis,DD iPSC-CMs have increased levels of ALDOA and PGAM2,two key players in glycolysis,as well as the myocardial hypertrophy-associated HSPA2;the baseline and the maximum capacity of glycolysis are increased in parallel with the cellular oxygen uptake,and the functions of lysosomes appear to change accordingly.From the results of the phosphoproteomic and electrophysiological data analyses,it can be concluded that DD iPSC-CMs have a reduced APA,an elevated MDP,and a decreasing calcium current density.Based on the prediction of kinase activity,CAMK2 activity may increase and affect the electrophysiological activities and contractility of cells by strengthening the phosphorylation of RyR2-S2808.