Razi Journal of Medical Sciences

fa اهمیت تمایز قلبی سلولهای بنیادی جنینی انسانی درشناسایی بیومارکرهای مرتبط با بیماریهای قلبی The Importance of Cardiac Differentiation of Human Embryonic Stem Cells in Identifying Biomarkers Associated with Heart Disease ژنتیک Genetic پژوهشي Research زمینه و هدف: توانایی سلول‌های بنیادی پرتوان برای تمایز به سمت رده قلبی، خصوصا در زمینه پزشکی بازساختی (Regenerative Medicine) توجه زیادی را به خود جلب کرده است. با این حال ، هدف نهایی برای ترمیم قلب در بیماری‌ها، چالش برانگیز است. سلول‌های قلبی مشتق شده از سلول‌های بنیادی که تا به امروز توصیف شده‌اند، ویژگی‌های عملکردی و ساختاری نسبتاً نابالغی را نشان می‌دهند. بنابراین توسعه استراتژی‌ها برای رسیدن به درجه بالاتری از بلوغ سلول‌های قلبی در شرایط in vitro اهمیت زیادی دارد. هدف از این مطالعه بررسی ژن‌های دارای تفاوت بیان در تمایز به سلول‌های قلبی و بررسی اهمیت آن‌ها در شناسایی بیومارکرهای بیماری‌های قلبی است. روش کار: ابتدا فایل‌های خام حاصل از RNA-sequencing نمونههای سلول‌های بنیادی و سلول‌های قلبی تمایز یافته از آن، از دیتاست با شماره GSE76523 بدست آمده و مراحل آنالیز نمونهها انجام شد. ژن‌های دارای تفاوت بیان در زبان برنامه نویسی R و با پکیج DESeq2 جداسازی شد. سپس، آنالیز عملکردی ژن‌ها از جمله مسیرهای سیگنالینگ، فرآیندهای بیولوژیکی و بیماری‌های قلبی و موقعیت‌های کروموزومی تأثیرپذیر از ژن‌های دارای تفاوت بیان در مسیر تمایز مورد بررسی قرار گرفت. یافته ها: نتایج نشان داد 1463ژن در بین نمونه‌های موردنظر دارای افزایش بیان و 1682 ژن نیز کاهش بیان داشتند. بعلاوه با بررسی آنالیزهای عملکردی مشخص شد ژن‌هایی مانند TTN، MYBPC3، TNNC1، TPM1، ACTC1، MYL2، TNNT2، MYH6 و MYH7 از جمله ژن‌هایی هستد که هم در تمایز سلول‌های قلبی و هم در کاردیومیوپاتی اتساعی و کاردیومیوپاتی هایپرتروفیک نقش خواهند داشت و همچنین موقعیت‌های کروموزومی chr5p11، chr4q، chr11q21 و chr11p1  به ترتیب جزء موقعیت‌های کروموزومی مهم در بین ژن‌های دارای تفاوت بیان بودند. نتیجه گیری: درنهایت، بررسیهای مولکولی دقیقتر منجر به دستیابی به ژن‌های  قابل اعتماد برای شناسایی بیومارکرهای قلبی خواهد شد که برای تحقیقات زیست پزشکی، توسعه دارو و کاربردهای بالینی ضروری است. Background & Aims: 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. Methods: 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. Results: 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. Conclusion: 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. تمایز سلولهای عضله قلبی, بیماریهای قلبی, سلولهای بنیادی Cardiomyocyte cells differentiation, Heart disease, Stem cells 153 165 http://rjms.iums.ac.ir/browse.php?a_code=A-10-5406-1&slc_lang=fa&sid=1 Akram Gholipour اکرم قلی پور akram.gholipour199069@gmail.com 3900319475328460083649 3900319475328460083649 No PhD Student, Department of Biology, Science and Research branch, Islamic Azad University, Tehran, Iran دانشجوی دکتری،گروه زیست شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران Farshad Shakerian فرشاد شاکریان farshadshakerian@gmail.com 3900319475328460083650 3900319475328460083650 No Professsor, Cardiogenetic Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran, & Cardiovascular Intervention Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran استاد، ﻣﺮﻛﺰ ﺗﺤﻘﯿﻘﺎت ﻗﻠﺐ و ﻋﺮوق ﺷﮭﯿﺪ رﺟﺎیﻲ، مرکز آموزشی، تحقیقاتی و درمانی قلب و عروق شهید رجایی، دانشگاه علوم پزشکی ایران، تهران، ایران، و ﻣﺮﻛﺰ ﺗﺤﻘﯿﻘﺎت ﻣﺪاﺧﻼت ﻗﻠﺒﻲ و ﻋﺮوﻗﻲ، مرکز آموزشی، تحقیقاتی و درمانی قلب و عروق شهید رجایی، دانشگاه علوم پزشکی ایران، تهران، ایران Ali Zahedmehr علی زاهدمهر arashzahedmehr@yahoo.com 3900319475328460083651 3900319475328460083651 No Associate Professsor, Cardiovascular Intervention Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran دانشیار، ﻣﺮﻛﺰ ﺗﺤﻘﯿﻘﺎت ﻣﺪاﺧﻼت ﻗﻠﺒﻲ و ﻋﺮوﻗﻲ، مرکز آموزشی، تحقیقاتی و درمانی قلب و عروق شهید رجایی، دانشگاه علوم پزشکی ایران، تهران، ایران Shiva Irani شیوا ایرانی Shi_irani@yahoo.com 3900319475328460083652 3900319475328460083652 No Associate Professsor, Department of Biology, Science and Research branch, Islamic Azad University, Tehran, Iran دانشیار، گروه زیست شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران Mahshid Malakootian مهشید ملکوتیان Malakootian@rhc.ac.ir 3900319475328460083653 3900319475328460083653 No Associate Professsor, Cardiogenetic Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran دانشیار، ﻣﺮﻛﺰ ﺗﺤﻘﯿﻘﺎت ﻗﻠﺐ و ﻋﺮوق ﺷﮭﯿﺪ رﺟﺎیﻲ، مرکز آموزشی، تحقیقاتی و درمانی قلب و عروق شهید رجایی، دانشگاه علوم پزشکی ایران، تهران، ایران Seyed Javad Mowla سیدجواد مولی sjmowla@yahoo.com 3900319475328460083654 3900319475328460083654 Yes Professsor, Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran استاد، دانشگاه تربیت مدرس، دانشکده علوم زیستی، گروه ژنتیک مولکولی، تهران، ایران