The Importance of Cardiac Differentiation of Human Embryonic Stem Cells in Identifying Biomarkers Associated with Heart Disease

An adult heart has limited regenerative potential, resulting in many problems such as ischemic heart disease and diseases in which heart muscle cells (cardiomyocytes) become defective, leading to heart failure. Since therapies for heart disease and heart transplants are limited due to the small number of heart donors, the ability of pluripotent stem cells to differentiate into heart disease, especially in regenerative medicine, has received considerable attention. Recently, stem cells can be induced to produce functional cardiomyocyte cells using a variety of methods (1-3). As a result, the formation of heart muscle cells from stem cells requires a deep understanding of the molecular processes involved in the evolution of myocardial cells(7). The aim of this study was to investigate genes with differential expression between embryonic stem cells and differentiated cardiomyocyte cells by bioinformatics analysis. Furthermore, we show the importance of these genes in the development of heart disease in order to utilize this model to achieve more appropriate biomarkers for heart diseases which are effective in both cardiac differentiation and heart disease.

RNA-sequencing samples of embryonic stem cells (hESC) and differentiated cardiomyocyte cells were obtained from the dataset number GSE76523 (14). FastQC software was used to check the quality of the raw data reads. Adapter sequences and low quality sequences were removed using Trimmomatic software, version 0.36. Using HISAT2 software, version 2.1.0, the sequences were aligned with the genome, and using the annotation reference obtained from the UCSC database and HTSeq software, version 0.9.1, the reading count of each gene was obtained and using DESeq2 Package in the R program. Genes with differential expression between stem cell samples and differentiated cells were isolated. To evaluate the functional analysis of genes with differences in expression between the two groups, the KEGG and Enrichr databases were used to examine important pathways and biological processes in which genes with differential expression were involved. In the ClinVar and DisGeNET databases, it was determined which genes with differential expression would play a role in heart diseases. In addition, using the database of chromosomal location, important chromosomal positions of genes with differential expression between the two groups were analyzed.

Our results showed that between the two groups, 1463 and 1682 genes had increased and decreased expression, respectively. Functional analysis and examination of disease databases demonstrated genes with differential expression had essential roles in dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), adrenergic signaling in myocardial cells, myocardial contraction, and myocardial infarction. Moreover, chr5p11, chr4q, chr11q21 and chr11p1 locations correspondingly were important chromosomal positions among genes with differential expression. Interestingly, it was found that genes involved in the differentiation of cardiomyocyte cells can also be involved in heart diseases as well. Among the genes with differential expression that were examined in different functional analyzes, genes including TTN, MYBPC3, TNNC1, TPM1, ACTC1, MYL2, TNNT2, MYH6, and MYH7 were important genes in both cardiac differentiation and heart diseases, especially different types of cardiomyopathies.

As the prevalence of cardiac disorders such as cardiomyopathies is increasing and due to the limitations of existing experimental models, suitable progress in treatment strategies for these disorders has not been encountered. One of the important factors in the development of cell-based models in heart disease is the existence of reliable methods for the production of heart cells from stem cells (17,18). In the present study, by investigating the signaling pathways and database, it was found that genes with differential expression in the differentiation of stem cells into heart cells can play a role in causing various heart diseases, especially dilated DCM and HCM. Better understanding of the underlying pathological mechanisms of these diseases will help prevent disease progression (24).In addition, we introduced genes which are involved in both cardiac cell differentiation and cardiomyopathy via bioinformatics analysis. Among them, TTN, MYBPC3, TNNC1, TPM1, ACTC1, MYL2, TNNT2, MYH6 and MYH7 genes were found to be involved in patients with dilated cardiomyopathy as well as hypertrophic cardiomyopathy. All of these genes were expressed in cardiac cells resulting from stem cell differentiation. Studies have shown that heterozygous mutations in the sarcomroponin T (TNNT2) protein, which were produced by induced stem cells, impaired calcium control and decreased contraction (27,28). Other stem cell models have examined the effect of LMNA encoding genes (LMNA A / C) and TTN on cardiomyopathy, and have found that TTN mutations are associated with heart disease, especially dilated cardiomyopathy (31). Other studies have shown sarcomere protein mutations, β-myosin heavy chain mutations, and MYH7 genes are associated with hypertrophic cardiomyopathy (32).In this disease stem cell models with mutations in MYH7 and MYBPC3, showed many of the features of the disease, such as cell enlargement, sarcomere disorder and contraction, as well as altered gene expression in calcium administration (33,34). Mutations in the TNNC1 gene also play a central role in the development of hypertrophic cardiomyopathy (38). A study of a large family with familial hypertrophic cardiomyopathy showed that a mutation in the TPM1 gene was associated with the clinical features of cardiac hypertrophy (39). Studies highlight an important role for MYL2 phosphorylation as an important contractile protein in the adult heart. These studies further show that the disappearance of mediated phosphorylation mechanisms in this gene causes dilated cardiomyopathy. Further studies have shown that in the model of hypertrophic cardiomyopathy using human cell-derived stem cells (hiPSC-CMs), the presence of cardiac actin E99K-ACTC1 mutation causes abnormal phenotypes in the produced hiPSC-CMs (40,41). In this study, important chromosomal positions including chr5p11, chr4q, chr11q21 and chr11p1 were introduced, which suggested that these positions may play a role in heart diseases, especially cardiomyopathies. Studies have not yet addressed the role of these chromosomal positions in heart diseases. Therefore, more detailed study and attention to tissue origin genes as well as studies of important chromosomal positions will be needed to achieve more specific biomarkers for the heart diseases and clarify the mechanisms of the heart diseases as well as cardiomyocyte differentiation.