Transcriptomic Characterization of Genes Regulating the Stemness in Porcine Atrial Cardiomyocytes during Primary In Vitro Culture

Heart failure remains a major cause of death worldwide. There is a need to establish new management options as current treatment is frequently suboptimal. Clinical approaches based on autologous stem cell transplant is potentially a good alternative. The heart was long considered an organ unable to regenerate and renew. However, several reports imply that it may possess modest intrinsic regenerative potential. To allow for detailed characterization of cell cultures, whole transcriptome profiling was performed after 0, 7, 15, and 30 days of in vitro cell cultures (IVC) from the right atrial appendage and right atrial wall utilizing microarray technology. In total, 4239 differentially expressed genes (DEGs) with ratio > abs |2| and adjusted p-value ≤ 0.05 for the right atrial wall and 4662 DEGs for the right atrial appendage were identified. It was shown that a subset of DEGs, which have demonstrated some regulation of expression levels with the duration of the cell culture, were enriched in the following GO BP (Gene Ontology Biological Process) terms: “stem cell population maintenance” and “stem cell proliferation”. The results were validated by RT-qPCR. The establishment and detailed characterization of in vitro culture of myocardial cells may be important for future applications of these cells in heart regeneration processes.

[1]  D. Bukowska,et al.  Transcriptomic Profile of Genes Regulating the Structural Organization of Porcine Atrial Cardiomyocytes during Primary In Vitro Culture , 2022, Genes.

[2]  Brad T. Sherman,et al.  DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update) , 2022, Nucleic Acids Res..

[3]  X. Qiu,et al.  PRRX1 Loss‐of‐Function Mutations Underlying Familial Atrial Fibrillation , 2021, Journal of the American Heart Association.

[4]  M. Goumans,et al.  Prrx1b restricts fibrosis and promotes Nrg1-dependent cardiomyocyte proliferation during zebrafish heart regeneration , 2021, Development.

[5]  C. Hodgkinson,et al.  A role for Sfrp2 in cardiomyogenesis in vivo , 2021, Proceedings of the National Academy of Sciences.

[6]  J. Uhm,et al.  A Variant Noncoding Region Regulates Prrx1 and Predisposes to Atrial Arrhythmias , 2021, Circulation research.

[7]  C. Hodgkinson,et al.  The role of Sfrp and DKK proteins in cardiomyocyte development , 2021, Physiological reports.

[8]  H. Arthur,et al.  Dynamic Changes in Endoglin Expression in the Developing Mouse Heart. , 2020, Gene expression patterns : GEP.

[9]  T. McKinsey,et al.  Faculty Opinions recommendation of BRD4 (Bromodomain-Containing Protein 4) Interacts with GATA4 (GATA Binding Protein 4) to Govern Mitochondrial Homeostasis in Adult Cardiomyocytes. , 2020, Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature.

[10]  Saptarsi M. Haldar,et al.  BRD4 (Bromodomain-Containing Protein 4) Interacts with GATA4 (GATA Binding Protein 4) to Govern Mitochondrial Homeostasis in Adult Cardiomyocytes , 2020 .

[11]  C. Dieterich,et al.  Updated and enhanced pig cardiac transcriptome based on long-read RNA sequencing and proteomics. , 2020, Journal of molecular and cellular cardiology.

[12]  Z. Zha,et al.  The multiple roles of Thy-1 in cell differentiation and regeneration. , 2020, Differentiation; research in biological diversity.

[13]  Nicolas F. Fernandez,et al.  Systems Analysis Implicates WAVE2 Complex in the Pathogenesis of Developmental Left-Sided Obstructive Heart Defects , 2020, JACC. Basic to translational science.

[14]  Yuehua Wu,et al.  Multiple Roles of sFRP2 in Cardiac Development and Cardiovascular Disease , 2020, International journal of biological sciences.

[15]  Xu Dong Zhang,et al.  Evaluating nuclear translocation of surface receptors: recommendations arising from analysis of CD44 , 2019, Histochemistry and Cell Biology.

[16]  E. Bober,et al.  Cardiomyocyte Sirt (Sirtuin) 7 Ameliorates Stress-Induced Cardiac Hypertrophy by Interacting With and Deacetylating GATA4. , 2019, Hypertension.

[17]  Astrid Gall,et al.  Ensembl 2020 , 2019, Nucleic Acids Res..

[18]  P. Pastuszko,et al.  Transcriptome profiling reveals activation of inflammation and apoptosis in the neonatal striatum after deep hypothermic circulatory arrest. , 2019, The Journal of thoracic and cardiovascular surgery.

[19]  Steven P Jones,et al.  A Physiological Biomimetic Culture System for Pig and Human Heart Slices. , 2019, Circulation research.

[20]  A. del Sol,et al.  Single-cell analysis of cardiogenesis reveals basis for organ level developmental defects , 2019, Nature.

[21]  I. Weissman,et al.  Regenerating the field of cardiovascular cell therapy , 2019, Nature Biotechnology.

[22]  J. Molkentin,et al.  Genetic Lineage Tracing of Sca-1+ Cells Reveals Endothelial but Not Myogenic Contribution to the Murine Heart , 2018, Circulation.

[23]  The Gene Ontology Consortium,et al.  The Gene Ontology Resource: 20 years and still GOing strong , 2018, Nucleic Acids Res..

[24]  S. Teichmann,et al.  Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement , 2018, Nature Communications.

[25]  P. He,et al.  Overexpression of Rac GTPase Activating Protein 1 Contributes to Proliferation of Cancer Cells by Reducing Hippo Signaling to Promote Cytokinesis. , 2018, Gastroenterology.

[26]  C. Hodgkinson,et al.  Insights from molecular signature of in vivo cardiac c-Kit(+) cells following cardiac injury and β-catenin inhibition. , 2018, Journal of molecular and cellular cardiology.

[27]  R. Hajjar,et al.  FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair , 2018, Circulation.

[28]  Reidar Andreson,et al.  Primer3_masker: integrating masking of template sequence with primer design software , 2018, Bioinform..

[29]  F. Prósper,et al.  Non-invasive in vivo imaging of cardiac stem/progenitor cell biodistribution and retention after intracoronary and intramyocardial delivery in a swine model of chronic ischemia reperfusion injury , 2017, Journal of Translational Medicine.

[30]  Linda T. Senbanjo,et al.  CD44: A Multifunctional Cell Surface Adhesion Receptor Is a Regulator of Progression and Metastasis of Cancer Cells , 2017, Front. Cell Dev. Biol..

[31]  T. Le,et al.  Cardiac progenitor cells for heart repair , 2016, Cell Death Discovery.

[32]  G. Fonarow,et al.  Epidemiology and aetiology of heart failure , 2016, Nature Reviews Cardiology.

[33]  N. Rosenthal,et al.  Revisiting Cardiac Cellular Composition. , 2016, Circulation research.

[34]  E. Marbán,et al.  Epigenomic Reprogramming of Adult Cardiomyocyte-Derived Cardiac Progenitor Cells , 2015, Scientific Reports.

[35]  Fátima Sánchez-Cabo,et al.  GOplot: an R package for visually combining expression data with functional analysis , 2015, Bioinform..

[36]  V. Dzau,et al.  Inhibition of Wnt6 by Sfrp2 regulates adult cardiac progenitor cell differentiation by differential modulation of Wnt pathways. , 2015, Journal of molecular and cellular cardiology.

[37]  Krzysztof Szade,et al.  Adult stem cells: hopes and hypes of regenerative medicine. , 2015, Acta biochimica Polonica.

[38]  Jens R. Nyengaard,et al.  Dynamics of Cell Generation and Turnover in the Human Heart , 2015, Cell.

[39]  B. Keene,et al.  Cardiac regenerative potential of cardiosphere-derived cells from adult dog hearts , 2015, Journal of cellular and molecular medicine.

[40]  James J. H. Chong,et al.  Cardiac stem cells: translation to human studies , 2015, Biophysical Reviews.

[41]  E. Braunwald The war against heart failure: the Lancet lecture , 2015, The Lancet.

[42]  Raphael Gottardo,et al.  Orchestrating high-throughput genomic analysis with Bioconductor , 2015, Nature Methods.

[43]  Y. Toiyama,et al.  RacGAP1 expression, increasing tumor malignant potential, as a predictive biomarker for lymph node metastasis and poor prognosis in colorectal cancer. , 2015, Carcinogenesis.

[44]  E. Marbán,et al.  Relative Roles of CD90 and c‐Kit to the Regenerative Efficacy of Cardiosphere‐Derived Cells in Humans and in a Mouse Model of Myocardial Infarction , 2014, Journal of the American Heart Association.

[45]  D. Torella,et al.  Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart , 2014, Nature Protocols.

[46]  Y. Kato,et al.  PRRX1 and PRRX2 distinctively participate in pituitary organogenesis and a cell-supply system , 2014, Cell and Tissue Research.

[47]  Milena B. Furtado,et al.  Cardiogenic Genes Expressed in Cardiac Fibroblasts Contribute to Heart Development and Repair , 2014, Circulation research.

[48]  J. Vieira,et al.  Re-activated adult epicardial progenitor cells are a heterogeneous population molecularly distinct from their embryonic counterparts. , 2014, Stem cells and development.

[49]  Chun-Keung Yu,et al.  Prostaglandin E2 promotes post-infarction cardiomyocyte replenishment by endogenous stem cells , 2014, EMBO molecular medicine.

[50]  I. Komuro,et al.  Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. , 2013, The Journal of Biological Chemistry.

[51]  Ciro Indolfi,et al.  Adult c-kitpos Cardiac Stem Cells Are Necessary and Sufficient for Functional Cardiac Regeneration and Repair , 2013, Cell.

[52]  C. Murry,et al.  Progenitor cells identified by PDGFR-alpha expression in the developing and diseased human heart. , 2013, Stem cells and development.

[53]  Fumihiro Sanada,et al.  Dissecting the Molecular Relationship Among Various Cardiogenic Progenitor Cells , 2013, Circulation research.

[54]  Y. Kaneda,et al.  The Transcription Factors Tbx18 and Wt1 Control the Epicardial Epithelial-Mesenchymal Transition through Bi-Directional Regulation of Slug in Murine Primary Epicardial Cells , 2013, PloS one.

[55]  E. Marbán,et al.  Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart , 2013, EMBO molecular medicine.

[56]  K. Clarke,et al.  Human Cardiosphere-Derived Cells from Patients with Chronic Ischaemic Heart Disease Can Be Routinely Expanded from Atrial but Not Epicardial Ventricular Biopsies , 2012, Journal of Cardiovascular Translational Research.

[57]  B. Faircloth,et al.  Primer3—new capabilities and interfaces , 2012, Nucleic acids research.

[58]  J. Epstein,et al.  Brief Ultrarapid Communication Islet1 Derivatives in the Heart Are of Both Neural Crest and Second Heart Field Origin Methods and Results: We Used an Intersectional Fate-mapping System Using the Rc::frepe Allele, Which Reports Dual Flpe and Cre Recombination. Combining Isl1 , 2022 .

[59]  Daniel Berman,et al.  Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial , 2012, The Lancet.

[60]  E. Marbán,et al.  Safety and Efficacy of Allogeneic Cell Therapy in Infarcted Rats Transplanted With Mismatched Cardiosphere-Derived Cells , 2012, Circulation.

[61]  N. S. Asli,et al.  Adult cardiac-resident MSC-like stem cells with a proepicardial origin. , 2011, Cell stem cell.

[62]  Marcus F Stoddard,et al.  Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial , 2011, The Lancet.

[63]  C. Indolfi,et al.  Endogenous cardiac stem cell activation by insulin-like growth factor-1/hepatocyte growth factor intracoronary injection fosters survival and regeneration of the infarcted pig heart. , 2011, Journal of the American College of Cardiology.

[64]  M. Pesce,et al.  C-kit+ cardiac progenitors exhibit mesenchymal markers and preferential cardiovascular commitment. , 2011, Cardiovascular research.

[65]  E. Marbán,et al.  Intramyocardial injection of autologous cardiospheres or cardiosphere-derived cells preserves function and minimizes adverse ventricular remodeling in pigs with heart failure post-myocardial infarction. , 2011, Journal of the American College of Cardiology.

[66]  Wei He,et al.  Exogenously administered secreted frizzled related protein 2 (Sfrp2) reduces fibrosis and improves cardiac function in a rat model of myocardial infarction , 2010, Proceedings of the National Academy of Sciences.

[67]  M. Roselle Abraham,et al.  Dedifferentiation and Proliferation of Mammalian Cardiomyocytes , 2010, PloS one.

[68]  B. Fleischmann,et al.  Cardiomyocyte cell cycle control and growth estimation in vivo--an analysis based on cardiomyocyte nuclei. , 2010, Cardiovascular research.

[69]  E. Morrisey,et al.  The Importance of Wnt Signaling in Cardiovascular Development , 2010, Pediatric Cardiology.

[70]  M. Fujita,et al.  Cyclin-dependent Kinase-9 Is a Component of the p300/GATA4 Complex Required for Phenylephrine-induced Hypertrophy in Cardiomyocytes* , 2010, The Journal of Biological Chemistry.

[71]  E. Marbán,et al.  Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue , 2009, PloS one.

[72]  C. Perou,et al.  Secreted frizzle-related protein 2 stimulates angiogenesis via a calcineurin/NFAT signaling pathway. , 2009, Cancer research.

[73]  Brad T. Sherman,et al.  Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists , 2008, Nucleic acids research.

[74]  Samuel Bernard,et al.  Evidence for Cardiomyocyte Renewal in Humans , 2008, Science.

[75]  Douglas A. Melton,et al.  In vivo reprogramming of adult pancreatic exocrine cells to β-cells , 2008, Nature.

[76]  M. Yacoub,et al.  Heterogeneic nature of adult cardiac side population cells. , 2008, Biochemical and biophysical research communications.

[77]  Thomas D. Schmittgen,et al.  Analyzing real-time PCR data by the comparative CT method , 2008, Nature Protocols.

[78]  P. Vliet,et al.  Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy , 2008, Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation.

[79]  Mark A Sussman,et al.  Evolution of the c‐kit‐Positive Cell Response to Pathological Challenge in the Myocardium , 2008, Stem cells.

[80]  Arjun Deb,et al.  SFRP2 Regulates Cardiomyogenic Differentiation by Inhibiting a Positive Transcriptional Autofeedback Loop of Wnt3a , 2008, Stem cells.

[81]  D. Atsma,et al.  Preservation of Left Ventricular Function and Attenuation of Remodeling After Transplantation of Human Epicardium-Derived Cells Into the Infarcted Mouse Heart , 2007, Circulation.

[82]  Jeffrey Robbins,et al.  Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury , 2007, Nature Medicine.

[83]  P. Couture,et al.  Thy-1 expression by cardiac fibroblasts: lack of association with myofibroblast contractile markers. , 2007, Journal of molecular and cellular cardiology.

[84]  Maido Remm,et al.  Enhancements and modifications of primer design program Primer3 , 2007, Bioinform..

[85]  E. Marbán,et al.  Regenerative Potential of Cardiosphere-Derived Cells Expanded From Percutaneous Endomyocardial Biopsy Specimens , 2007, Circulation.

[86]  D. Atsma,et al.  Epicardial Cells of Human Adults Can Undergo an Epithelial‐to‐Mesenchymal Transition and Obtain Characteristics of Smooth Muscle Cells In Vitro , 2007, Stem cells.

[87]  Yunfu Sun,et al.  Multipotent Embryonic Isl1 + Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification , 2006, Cell.

[88]  S. Ikeda,et al.  Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure , 2006, Proceedings of the National Academy of Sciences.

[89]  P. Chomczyński,et al.  The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on , 2006, Nature Protocols.

[90]  K. Ando,et al.  Expression of CD90 on keratinocyte stem/progenitor cells , 2006, The British journal of dermatology.

[91]  G. Fang,et al.  MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[92]  F. Mouquet,et al.  CD31− but Not CD31+ Cardiac Side Population Cells Exhibit Functional Cardiomyogenic Differentiation , 2005, Circulation research.

[93]  Karl-Ludwig Laugwitz,et al.  Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages , 2005, Nature.

[94]  Giulio Cossu,et al.  Isolation and Expansion of Adult Cardiac Stem Cells From Human and Murine Heart , 2004, Circulation research.

[95]  M. Goumans,et al.  Endoglin promotes endothelial cell proliferation and TGF‐β/ALK1 signal transduction , 2004, The EMBO journal.

[96]  Daniel J Garry,et al.  Transcriptional Regulation of Cardiac Progenitor Cell Populations , 2004, Circulation research.

[97]  Michael D. Schneider,et al.  Cardiac progenitor cells from adult myocardium: Homing, differentiation, and fusion after infarction , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[98]  D. Torella,et al.  Adult Cardiac Stem Cells Are Multipotent and Support Myocardial Regeneration , 2003, Cell.

[99]  I. Komuro,et al.  Roles of cardiac transcription factors in cardiac hypertrophy. , 2003, Circulation research.

[100]  D. Bodine,et al.  Bone marrow stem cells regenerate infarcted myocardium , 2003, Pediatric transplantation.

[101]  M. Rudnicki,et al.  The post‐natal heart contains a myocardial stem cell population , 2002, FEBS letters.

[102]  E. Fearon,et al.  The SLUG zinc-finger protein represses E-cadherin in breast cancer. , 2002, Cancer research.

[103]  M. Nieto,et al.  The snail superfamily of zinc-finger transcription factors , 2002, Nature Reviews Molecular Cell Biology.

[104]  T. Kitamura,et al.  MgcRacGAP Is Involved in Cytokinesis through Associating with Mitotic Spindle and Midbody* , 2001, The Journal of Biological Chemistry.

[105]  Y. Ikeda,et al.  MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells. , 2000, Blood.

[106]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[107]  M. DeRuiter,et al.  Patterns of paired‐related homeobox genes PRX1 and PRX2 suggest involvement in matrix modulation in the developing chick vascular system , 1998, Developmental dynamics : an official publication of the American Association of Anatomists.

[108]  Adriana C. Gittenberger-de Groot,et al.  Expression patterns of the paired-related homeobox genes MHox/Prx1 and S8/Prx2 suggest roles in development of the heart and the forebrain , 1995, Mechanisms of Development.

[109]  D G Wilkinson,et al.  Control of cell behavior during vertebrate development by Slug, a zinc finger gene. , 1994, Science.

[110]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[111]  D. Torella,et al.  Heterogeneity of Adult Cardiac Stem Cells. , 2019, Advances in experimental medicine and biology.

[112]  G. Fonarow,et al.  2017 ACC/AHA/HFSA Focused Update of the 2013 ACCF/AHA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America. , 2017, Circulation.

[113]  T. Weiss,et al.  Thy-1 (CD90)-Positive Hepatic Progenitor Cells, Hepatoctyes, and Non-parenchymal Liver Cells Isolated from Human Livers. , 2017, Methods in molecular biology.

[114]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[115]  Biykem Bozkurt,et al.  2013 ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. , 2013, Circulation.

[116]  R. Wirtz,et al.  Validity of the proliferation markers Ki67, TOP2A, and RacGAP1 in molecular subgroups of breast cancer , 2012, Breast Cancer Research and Treatment.

[117]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[118]  D. Prockop,et al.  Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. , 2006, Cytotherapy.

[119]  D. Marchuk,et al.  Endoglin, an ancillary TGFbeta receptor, is required for extraembryonic angiogenesis and plays a key role in heart development. , 2000, Developmental biology.

[120]  Thomas D. Schmittgen,et al.  Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 2 DD C T Method , 2022 .